c-Jun-mediated repression and transactivation of fibronectin.

Fibronectin (FN) is transactivated by human papillomavirus type 16 (HPV16) E6 via the induction of c-Jun-ATF-2 complexes binding to the cyclic AMP response element (CRE) in the FN promoter. The present study analyzed c-Jun regulation of FN gene expression. Northern and immunoblot analyses showed that c-Jun expression was enhanced in HPV16-E6-expressing cells. However, mouse 10T1/2 cell lines overexpressing c-Jun showed an inverse correlation between the expression levels of c-Jun and those of FN. Luciferase assays indicated that the FN promoter was strongly repressed in c-Jun-overexpressing mouse 10T1/2 cells. Deletion and mutation analyses of the FN promoter revealed that repression of the FN promoter by c-Jun depends on the CRE located at -160 relative to the start site of transcription. Supershift assays of CRE-bound complexes from HPV16-E6-expressing and c-Jun-overexpressing cells suggested that the presence of ATF-2 in the complexes binding to CRE was required for the transactivation of the FN gene. Collectively, our study suggests that the FN gene can be either transactivated or repressed by c-Jun depending on the cell context.

Oncolytic viral therapy for cervical and ovarian cancer cells by Sindbis virus AR339 strain.

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.

Effects of adenoviral vector-mediated BDNF expression on the bulbectomy-induced apoptosis of olfactory receptor neurons.

The expression of adenoviral vector (Ad)-mediated lacZ and brain-derived neurotrophic factor (BDNF) in mouse olfactory epithelium (OE) was examined, and the effect of BDNF on the survival of the bulbectomized OE was evaluated. A recombinant adenovirus, Ax1CAlacZ, was administrated into the mouse OE after bulbectomy, and the expression of a transferred E. coli beta-galactosidase (beta-gal) gene was confirmed by X-gal staining. The expression and effects of exogenous BDNF in the OE after bulbectomy were examined using immunohistochemistry and the TUNEL method. The adenoviral vector-mediated expression of beta-gal in the mouse OE was detectable for up to 14 days after bulbectomy in vivo. The Ad-mediated expression of BDNF was also observed in the OE after bulbectomy. Exogenously induced BDNF suppressed the degenerative changes of bulbectomized OE. TUNEL staining indicated that the exogenous BDNF enhanced the survival of the bulbectomized OE by inhibiting apoptosis. Ad-mediated expression of BDNF in the mouse nasal mucosa alleviated degenerative changes in bulbectomized OE. Ad-mediated transfer of neurotrophic factors might be applicable in the treatment of olfactory disorders.

FAK promotes organization of fibronectin matrix and fibrillar adhesions.

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.

Survival regulation in pancreatic cancer cells by c-Jun.

Over 90% of human pancreatic cancers harbor an activating point mutation in the K-ras gene at codon 12. However, it is not clear whether all downstream K-ras are activated and which downstream contributes to the cell survival and proliferation of pancreatic cancer cells. MEK kinase 1 (MEKK1)-c-Jun N-terminal kinase (JNK)-c-Jun pathway has an important role in cell proliferation, survival and apoptosis in various cells. We previously demonstrated that the dominant negative form of MEKK1 (DN-MEKK) inhibits the survival of human pancreatic cancer cell lines. In this study we investigated whether JNK-c-Jun, the downstream pathway of DN-MEKK, affects the survival of human pancreatic cancer cell lines. Colony formation assays indicated that c-Jun failed to inhibit the survival of pancreatic cancer cells, whereas c-Jun remarkably inhibited the cell survival of non-pancreatic cancer cells. Reporter gene assays using Gal4-c-Jun and gel retardation assays indicated that c-Jun functions were activated in growing pancreatic cancer cells. These results revealed that c-Jun activation does not prevent the cell survival of pancreatic cancer cells in contrast to non-pancreatic cancer cells. It appears that MEKK1-JNK-c-Jun pathway fails to act as a negative regulator for the cell survival of pancreatic cancer cells. Greater understanding of these mechanisms may be helpful in the treatment of pancreatic cancer.

Vascular endothelial growth factor and the risk of smoking-related COPD.

STUDY OBJECTIVES: Vascular endothelial growth factor (VEGF) signaling may be required for maintenance of the alveolar structures, and alveolar septal cell apoptosis could contribute to the pathogenesis of COPD presenting emphysematous changes; however, the common mutation at position 936 in the 3' untranslated region of the VEGF gene, a C to T substitution (the C allele was denoted as 1, and the T allele as 2), VEGF936*2, has been reported to be associated with significantly lower VEGF plasma levels. Based on these concepts, we hypothesized that VEGF936*1/2 polymorphism may be linked to the development of COPD. DESIGN: The differences in VEGF936*1/2 allele frequency were examined in 113 patients with smoking-related COPD and two control groups (101 smoker/ex-smoker control subjects and 102 population control subjects) using the polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: VEGF936*1/2 allele frequencies did not differ among the groups: 0.792/0.208 in COPD patients, 0.822/0.178 in smoker/ex-smoker control subjects, and 0.842/0.152 in population control subjects. CONCLUSION: The 936 C/T polymorphism of the VEGF gene (including both homozygous and heterozygous) was not associated with the development of COPD (odds ratio, 1.23; 95% confidence interval, 0.760 to 1.995).

Dominant negative MEKK1 inhibits survival of pancreatic cancer cells.

Human pancreatic cancers harbor mutations in the K-ras gene, and these mutations convert the gene oncogenic and constitutively active forms. However, in pancreatic cancer cells little is known about the activation of the downstream pathways of Ras, MEK-ERK and MEKK1-JNK, and their roles in cell survival and proliferation. An analysis of nine pancreatic cancer tissues revealed JNK activation in all tumor samples and ERK activation in three tumor samples. Colony formation assays by transfection of dominant negative mutants of Ras, ERK or MEKK1 into pancreatic cancer cell lines (BxPC-3, PANC-1, MIAPaCa-2 and AsPC-1) and an amnion-derived cell line (FL) revealed that DN-MEKK strongly inhibits the survival of colonies in pancreatic cancer cells, but not in FL cells. In vitro kinase assays and luciferase assays using the Gal4c-Jun system revealed that in pancreatic cancer cells DN-MEKK fails to inhibit JNK activation. In PANC-1 cells, c-Jun was found to be a major component of protein component binding to AP-1 site and CRE, but not in FL cells. The inhibitory effect of DN-MEKK in PANC-1 cells was thought to be the result of the inhibition of c-Jun DNA-binding. The difference of suppression in pancreatic cancer cells and non-pancreatic cancer cells suggested that the MEKK1 pathway mainly contributes to cell survival in pancreatic cancer cells and may provide an advantage for the gene therapy of pancreatic cancers using DN-MEKK expression vectors.

Association of tumor necrosis factor-alpha gene promoter polymorphism with low attenuation areas on high-resolution CT in patients with COPD.

STUDY OBJECTIVES: Alveolar septal cell apoptosis may contribute to the pathogenesis of emphysema. Because tumor necrosis factor (TNF)-alpha is assumed to play an important role in the induction of apoptosis, and allele 2 of the polymorphism at position--308 in the promoter of the TNF-alpha gene has been associated with alteration of TNF-alpha secretion in vitro, we hypothesized that genotypes containing this allele would show more destructive emphysematous changes of the lung. DESIGN: The percentage ratio of the low attenuation area to the corresponding lung area was evaluated using a visual scoring system for CT findings in patients with COPD (n = 84), and these patients were classified into two groups: those with a visual score < 11 and those with a visual score > or = 11. A polymerase chain reaction-based assay was developed to determine the TNF-alpha genotype (TNF-alpha-308*1/2) between subjects with high and low visual scores on chest CT scans. RESULTS: The TNF-alpha-308*1/2 allele frequency tended to differ between patients with a visual score < 11 (0.90/0.10) and those with a visual score > or = 11 (0.81/0.19) [odds ratio, 2.15; 95% confidence interval, 0.87 to 5.30; p = 0.09]. CONCLUSION: These results indicate that the TNF-alpha-308 allele 2 may be partly associated with the extent of emphysematous changes in patients with COPD.

MEKK1 induces c-Jun complexes that act as negative regulators for cell survival and proliferation of HCC cells.

c-Jun has a variety of functions including proliferation, differentiation and death. c-Jun is specifically phosphorylated by c-Jun N-terminal kinase (JNK) which is regulated by Ras-MEKK1-MKK4/7 pathway. Previous studies showed that c-Jun protein plays a positive role in cell proliferation of normal hepatocytes and was detected in hepatocellular carcinoma (HCC) tissues. However, the function of c-Jun in HCC cells has not been examined. The aim of this study was to investigate whether the MEKK1-JNK signaling pathway and c-Jun may be involved in the survival and proliferation of HCC. Surprisingly, an active not dominant negative form of MEKK1 (CA-MEKK1) remarkably inhibited the colony formation of HCC cells. Gel retardation assays indicated that CA-MEKK1 induces c-Jun DNA binding, and luciferase assays exhibited that CA-MEKK1 enhances the transactivating activity of c-Jun in HCC cells. These results suggested that the inhibitory effect of CA-MEKK1 on colony formation is likely to be mediated by c-Jun. As expected, when wild-type c-Jun was transfected, the colony formation was significantly reduced. Especially in HuH7 cells, c-Jun transfected cells failed to make any colonies. Our data suggested that c-Jun activation can induce negative effect on survival and proliferation of HCC cells.

HPV 16-E6-mediated degradation of intrinsic p53 is compensated by upregulation of p53 gene expression in normal cervical keratinocytes.

In this study we investigated the relationship between the development of either HSILs (high-grade squamous intraepithelial lesions) or invasive cancers of the uterine cervix and the p53 codon 72 polymorphisms consisting of arginine (Arg)- or proline (Pro)-encoded allele in Japanese populations. Furthermore, we determined if intrinsic p53 is affected by the p53-degradation activities of human papillomavirus type 16 (HPV 16)-E6 in normal cervical keratinocytes with each p53 codon 72 genotype. Using PCR with p53 codon 72 polymorphic allele-specific primers, p53 genotypes of 112 normal cervical specimens and 88 cervical lesions including 44 HSILs and 44 invasive cancers were determined. The expression levels of p53 proteins and mRNAs in keratinocytes following the expression of HPV 16-E6 from an HPV 16-E6-expressing recombinant adenovirus were analyzed. The frequencies of p53 genotypes, Pro/Pro, Arg/Pro and Arg/Arg were, respectively, 12, 49 and 39% in controls; and 18, 55 and 27% in HSILs; and 11, 41 and 48% in invasive cancers. The expression levels of the p53 proteins in normal human keratinocytes of each p53 codon 72 genotype were not affected by the introduction of HPV 16-E6, whereas the p53 mRNAs were found to be upregulated regardless of the p53 genotypes when HPV 16-E6 was expressed. In statistical analysis, no relationships between p53 genotypes and the development of cervical neoplasias were found. Furthermore, we demonstrated that p53 protein expression levels in normal cervical keratinocytes is not affected by the p53-degradation activities of HPV E6, probably due to a tight transcriptional regulation of p53.

Expression of the potential novel gene E6DG1 downregulated by the E6 protein of human papillomavirus type 16 is correlated with anchorage-independent growth.

A novel gene transcript that is downregulated by HPV16 E6 protein was identified in mouse cells using differential hybridization and designated E6DG1. The cloned cDNA of E6DG1 was 1.3 kb in length and contained a small ORF potentially encoding a polypeptide of 45 amino acids. In vitro transcription and translation of E6DG1 cDNA resulted in a product of approximately 7 kDa and Western blot analysis using antibodies for E6DG1 peptide detected 7 and 14 kDa proteins. Downregulation of E6DG1 mRNA levels in cells expressing HPV16 E6 protein was observed at subconfluent cell densities, but not in confluent cells. Repression of E6DG1 protein enhanced the anchorage-independent growth and weakened the cell adhesion in Panc1 cells. Immunofluorescence analysis revealed the localization of E6DG1 within the nucleus. These results indicate that the E6DG1 protein may function in a signaling pathway related to anchorage-independent growth and adhesion control.

Alteration of integrin expression relates to malignant progression of human papillomavirus-immortalized esophageal keratinocytes.

To investigate cellular changes related to the malignant progression of keratinocytes, we studied the serum-resistant clones from CHEK-1, a human papillomavirus type 16 E6/E7-immortalized esophageal cell line cultured in a serum-free medium. Established clones exhibited morphologic variety. Slow growing clones presented in cuboidal shapes with tight cellular adhesion and highly expressed alpha2 and alpha6beta4 integrins. Moderately proliferating clones showed loose intercellular adhesion and reduced expression of alpha2 integrin. Spindle-shaped, rapidly proliferating clones with prominent actin stress fibers demonstrated reduced alpha6 and alpha4 integrin expression in addition to alpha2 integrin and showed anchorage-independent growth. Reduced expression of alpha2 integrin was observed between 50 and 100 population doubling lengths (PDLs) during the immortalization of CHEK-1. These results suggest that the reductions of alpha2 and alpha6beta4 integrins are related to changes seen during immortalization and malignant progression.