Jun Kusaka

Effects of Protein Tyrosine Kinase Inhibitors on the Replication of Herpes Simplex Virus and the Phosphorylation of Viral Proteins

The effect of protein tyrosine kinase (PTK) inhibitors on the replication of herpes simplex virus (HSV) was examined. Tyrphostins AG17, AG213, AG490, and AG555, and herbimycin A all inhibited the plaque formation of HSV type 1 (HSV-1) in Vero cells, but AG17, AG490, and AG555 exhibited a more selective antiviral effect. In the presence of 0.4 microM AG17, the virus production 24 h after infection was decreased to 7.7% of the untreated control level. Even if the treatment was started 12 h after the initiation of infection, the viral titer was reduced by 82.4%, compared with the untreated control level. In HSV-1-infected cells ICPs 6, 17/18, 19/20, and 25 were tyrosine-phosphorylated proteins. The synthesis and phosphorylation of these proteins were inhibited by AG17, and suppression of ICP 19/20, which were identified as the UL47 gene products, was the greatest. In contrast, the in vitro autophosphorylation of viral proteins was not affected by this PTK inhibitor. These results indicate that tyrphostin may represent a novel class of inhibitors of HSV-1, and that the viral proteins which have phosphorylated tyrosine residues and are suppressed by AG17 most significantly are the products of the UL47 gene, the tegument proteins VP13/14.

Germline mutations of the PTCH gene in Japanese patients with nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental and skeletal anomalies, palmo-plantar pits, odontogenic keratocysts, ectopic calcification, and occurrence of various types of tumors including basal cell carcinoma. Recent evidence has indicated that the human homologue of a Drosophila segment polarity gene, PTCH, is a NBCCS susceptibility gene. In the study presented here, we detected two novel mutations of the PTCH gene, I805X/2395delC and Y93X/C297A, in two unrelated Japanese patients. Early protection of the skin from the sunlight is important to the prevention of BCC development in NBCCS patients. Genetic analysis of the PTCH gene is essential for the early, definitive diagnosis of NBCCS, especially before the expression of clinical manifestations is complete.

Effect of local administration of epidermal growth factor on 9,10-dimethyl-1,2-benzanthracene-induced tumour formation in hamster cheek pouch

The effect of local administration of epidermal growth factor (EGF) on 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tumour formation was investigated in a hamster cheek pouch carcinogenesis model. DMBA-treated hamsters underwent either sialoadenectomy (groups 1 and 2) or a sham operation (groups 3 and 4). Thereafter, EGF (groups 1 and 3) or vehicle (groups 2 and 4) was applied to the cheek pouches for 6 weeks. Fourteen weeks after the beginning of the experiment, the number of cheek pouch tumours was significantly greater in EGF-treated hamsters than in vehicle-treated hamsters, irrespective of whether the submandibular glands had been removed. The number of forestomach tumours, induced by DMBA application to the cheek pouches, was also increased by EGF. These results suggest that EGF applied from the luminal side of the mucosa stimulates tumour formation in the hamster cheek pouch and forestomach.

Inhibitory effect of tyrphostin on the replication of herpes simplex virus type 1.

SummaryTyrphostins 9 and 47, inhibitors of protein-tyrosine kinase, inhibited the replication of herpes simplex virus type 1 (HSV-1), whereas tyrphostin 1, which does not inhibit protein-tyrosine kinase, did not affect the replication of HSV-1. The inhibitory effect of tyrphostin 9 was more potent than that of tyrphostin 47, and the IC50 of tyrphostin 9 was 40 nM. Sodium orthovanadate, an inhibitor of protein phosphotyrosine phosphatase, increased HSV-1 plaque formation and its effect was partly reversed by tyrphostin 9. The phosphorylation of viral phosphoproteins was decreased by tyrphostin 9 in a dose-dependent manner, but the tyrphostin 9-induced reduction of protein synthesis was not dose-dependent. At the late stage of infection, tyrosine phosphorylation was demonstrated in HSV-1 phosphoproteins. These results indicate that protein-tyrosine kinase is involved in the replication of HSV-1 and that tyrphostin can inhibit the synthesis and post-translational phosphorylation of the viral proteins.

Enhancement of herpes simplex virus-induced polykaryocyte formation by 12-O-tetradecanoyl phorbol 13-acetate: association with the reorganization of actin filaments and cell motility.

A morphological change induced by syn– herpes simplex virus type 1 (HSV-1), polykaryocyte formation, was enhanced by treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) in A431 cells. TPA treatment decreased the number of stress fibers, but led to the development of spike-like filopodia and actin-containing long projections. Similar reorganization of actin filaments was observed in HSV-1-induced polykaryocytes. The actin filament-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, inhibited the effect of TPA on polykaryocyte formation, indicating that the actin microfilament system plays a key role in this event. HSV-1 glycoprotein D (gD) was present in the cytoplasm of HSV-1-infected cells and gD gene-transfected cells; its expression became prominent at long cell projections in the presence of TPA. These findings suggest that the reorganization of actin filaments and cell motility are associated with the enhancing effect of TPA on HSV-1-induced polykaryocyte formation.

Effect of Ca2+-dependent cell death on the release of herpes simplex virus

Summary. The effect of a variety of cell death-inducing reagents on the release of herpes simplex virus type 1 (HSV-1) was examined. Ionomycin was found to increase the release of HSV-1, whereas no significant increase was observed by the treatment with TNF-α, anti-Fas antibody, C2-ceramide, sphingosine, H-7, tyrphostin and camptothecin. Ionomycin induced an immediate early peak and a subsequent long-lasting elevation of intracellular Ca2+ concentration ([Ca2+]i). In the absence of extracellular Ca2+, ionomycin neither elevated [Ca2+]i nor increased the release of HSV-1 from the infected cells, indicating that Ca2+ influx play an important role in the release of HSV-1. Studies with trypan blue and annexin V staining revealed that the ionomycin-induced alteration of [Ca2+]i was accompanied by cell death of the infected cells. Disintegration of cell membrane, cytoplasmic vacuole formation and the leakage of virus particles from the cell surface were observed by electron microscopy. These results indicate that Ca2+-dependent cell death showing necrotic alteration is responsible for the enhanced release of HSV-1. The data also give some initial insights into the functional importance of cell death during the late stages of HSV-1 infection.

Hexamethylene bisacetamide as a chemopreventive agent in hamster cheek pouch tumorigenesis

The chemopreventive effect of oral and intraperitoneal (i.p.) administration of hexamethylene bisacetamide (HMBA) on 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tumour formation in hamster cheek pouches was investigated. Male Syrian hamsters were treated by painting both cheek pouches with a 0.5% solution of DMBA twice weekly for 11 weeks. In addition to DMBA application, Group 1 hamsters were given 1% HMBA continuously in the drinking water and Group 2 hamsters received i.p. injection of HMBA at a dose of 500 mg/kg three times per week during the experiment. Group 3 animals received DMBA application alone. Thirteen weeks after the start of the experiment, the numbers of cheek pouch tumours and tumour volume were significantly decreased by oral but not i.p. administration of HMBA. Low levels of HMBA were detected in the plasma of the hamsters which were given 1% HMBA in drinking water. These results indicate that oral administration of HMBA can act as a chemopreventive agent against hamster cheek pouch tumorigenesis.