Yuji Shino

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.

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.

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.

Dynamic changes in AP-1 transcription factor DNA binding activity in rat brain following administration of antidepressant amitriptyline and brain-derived neurotrophic factor.

The present study was undertaken to examine the effects of the antidepressant, amitriptyline, and brain-derived neurotrophic factor (BDNF) on activator protein-1 (AP-1) DNA binding activity in the rat brain. Acute administration of amitriptyline (5 or 10 mg/kg) initially increased but then decreased AP-1 DNA binding activity in the rat frontal cortex and hippocampus. Chronic administration of amitriptyline (5 or 10 mg/kg, once daily for 3 weeks) initially decreased AP-1 DNA binding activity but ultimately resulted in its persistent elevation in the rat frontal cortex. In contrast, the chronic administration of amitriptyline did not affect the low activity of AP-1 DNA binding in the hippocampus. However, chronic administration of amitriptyline (10 mg/kg, once daily for 3 weeks) significantly increased BDNF protein levels in the hippocampus (by 26.9%) and frontal cortex (by 24.6%). Direct infusion of BDNF (1 microg) into the hippocampal dentate gyrus significantly increased hippocampal AP-1 DNA binding activity. These results suggest that AP-1 transcription factor may be modulated by BDNF and that it may be an important target for the action of antidepressants.

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.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells.

Background:Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells.Methods:We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs).Results:Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase–mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IκBα was associated with SIN-induced apoptosis.Conclusion:As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells.

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.

Cloning and sequencing of the murine farnesyltransferase α-encoding cDNA from a cell line which expresses the human papillomavirus type-16 E6 gene

Using a differential hybridization technique, the murine farnesyltransferase alpha (FTA)-encoding cDNA was cloned from a mouse 10T1/2 cell line which expresses the human papillomavirus type 16 (HPV16) E6 gene. Sequence analysis revealed that the murine 1647-bp FTA cDNA encoded 377 amino acid (aa). The murine and human sequences showed 83.2% nucleotide and 92.6% aa sequence identity.