Hiroki Iga

Therapy for oral squamous cell carcinoma by tegafur and streptococcal agent OK-432 in combination with radiotherapy: Association of the therapeutic effect with differentiation and apoptosis in the cancer cells

Twenty patients with oral squamous cell carcinoma having mainly stage II or III lesions without distant metastasis, were treated with tegafur and streptococcal agent, OK-432, in combination with radiotherapy. As a consequence, 16 cases among the treated 20 cases showed complete remission by this therapy alone. Especially, we have found that the squamous cell carcinoma arising in non-keratinizing oral epithelium rather than in keratinizing oral epithelium has better response to this therapy. Among the 16 cases with complete remission (CR) by the current therapy, 10 cases were histopathologically diagnosed as well-differentiated squamous cell carcinoma and six cases as moderately differentiated squamous cell carcinoma. When we examined immunohistochemically the expres-sion of various antigens such as proliferating cell nuclear antigen (PCNA), p53 and LeY or the presence of DNA fragmentation by nick-end labelling in the biopsy materials taken at the first visit to our clinic from 20 patients treated with the current therapy, the CR group showed a significantly increased LeY expres-sion level ( p< 0.05) and DNA fragmentation rate ( p< 0.05) as compared with the partial response (PR, n= 3) + no change (NC, n= 1) group. On the other hand, the CR group with respect to PCNA expression level was significantly decreased as compared with the PR + NC group ( p< 0.05). From these findings, it can be considered that the therapy for oral squamous cell carcinoma by UFT and OK-432 in combination with radiotherapy is very effective, which may be associated with differentiation or apoptosis in oral squamous carcinoma cells. In addition, we present the clinical findings and results of immunohistochemical staining for the biopsy materials obtained from four CR cases treated with the current therapeutic method.

The treatment with differentiation- and apoptosis-inducing agent, vesnarinone, of a patient with oral squamous cell carcinoma

A patient with histopathological recurrent oral cancer with well-differentiated squamous cell carcinoma, was treated with differentiation- and apoptosis-inducing agent, vesnarinone, per os at a dose of 180 mg/day for 56 days and then at a dose of 60 mg/day for 93 days. The vesnarinone administration caused complete remission of the tumour. It has been found by immunohistochemical staining and PCR-SSCP analysis that the recurrent tumour has wild type p53 gene and relative high level of LeY expression as well as DNA fragmentation in the cancer cells, as assessed by nick-end labelling. These findings suggest that the cure of oral squamous cell carcinoma observed in this case might be associated with induction of differentiation and apoptosis of cancer cells by vesnarinone.

Macromolecular synthesis at the early stage of herpes simplex virus type 2 (HSV-2) latency in a human neuroblastoma cell line IMR-32: repression of late viral polypeptide synthesis and accumulation of cellular heat-shock proteins

SummaryWe have shown that a latent infection of herpes simplex virus type 2 (HSV-2) can be established in a human neuroblastoma cell line IMR-32 if the infected cells are cultured at 40°C. In the present study, viral polypeptides and cellular heat-shock proteins which were synthesized in HSV-2 infected IMR-32 cells cultured at 40°C were analyzed by polyacrylamide gel electrophoresis. It was found that the synthesis of late viral polypeptide ICP 5 was markedly reduced in the infected cells at 40°C as compared with those at 37°C. Although infection of IMR-32 cells with HSV-2 at 40°C resulted in shutoff of cellular protein synthesis, it was found that some cellular heat-shock proteins (90, 72 and 70 kd polypeptides) were synthesized and accumulated intracellularly. These findings suggest that modification of cascade regulation of HSV-2 polypeptide synthesis and/or accumulation of heat-shock proteins may be involved in the incomplete arrest of virus growth and in survival of the infected cells, leading to the establishment of HSV-2 latency in IMR-32 cells.

The role of epithelial cell differentiation in the expression of herpes simplex virus type 1 in normal human oral mucosa in culture

SummaryWe have examined by immunofluorescent antibody staining technique the expression of herpes simplex virus type 1 (HSV-1) in organ cultures of the normal human oral mucosa. The expression of HSV-1 antigen was found selectively in the epithelial cell layers in relatively undifferentiated states such as basal layer and lower prickle cell layer in addition to the basement membrane. When the epithelial cells dissociated from the oral mucosa were infected with HSV-1 and association of the HSV-1 expression with the cellular differentiation was examined, the epithelial cells containing laminin in an undifferentiated state were permissive for the expression of HSV-1 antigen whereas terminally differentiated epithelial cells with the cornified envelope did not express HSV-1 antigen. These findings indicate that the expression of HSV-1 antigen is restricted in the mucosal epithelial cells in a differentiated state, although the possibility that the cornified envelope might protect the cells from infection is not excluded.

A latent infection of herpes simplex virus type 2 in a human neuroblastoma cell line IMR-32.

SummaryHuman neuroblastoma (IMR-32) cells were infected with herpes simplex virus type 2 (HSV-2) at a multiplicity of infection (MOI) of 2 plaque-forming units (PFU)/cell and were cultured at 40° C for 14 days. Then neither infectious virus particles nor virus capsids were detected in these cells whereas the presence of virus-specific antigens was observed by immunofluorescent antibody staining technique in 16.9±3.2 per cent of the infected cell population. When the cultivation temperature was shifted down from 40° C to 35° C, reactivation of virus growth occurred after lag periods of 2–9 days. These findings indicate that the IMR-32 cells can be latently infected with HSV-2 at 40° C and that virus growth may be inhibited at the level of synthesis of virus-specific macromolecules or at some step preceding nucleocapsid formation.

Emergence of osteoblast-like cells in a neoplastic human salivary cancer cell line after treatment with 22-oxa-1α, 25-dihydroxyvitamin D3

A neoplastic clonal cell line, which was prepared by 5-azacytidine treatment of a neoplastic human salivary intercalated duct cell line, was cultivated in the presence of 22-oxa-1alpha, 25-dihydroxyvitamin D3 and 3 mM beta-glycerophosphate. Major alterations, such as expression of type I collagen and alkaline phosphatase as well as of human osteopontin and osteonectin, were observed in these cells with a phenotype similar to osteoblasts. In addition, formation of bone nodule was observed in the cultured cells. The tumors produced by transplantation into nude mice of the clonal cells were treated with 22-oxa-1alpha, 25-dihydroxyvitamin D3 and examined for tumor growth and morphology. Consequently, growth of the treated tumor was significantly suppressed. Moreover, it was found that bone formation was induced in the treated tumor, in which the tumor cells around bone formation expressed human osteopontin and osteonectin mRNA as could be detected by in situ hybridization. The above findings indicate that the emergence of osteoblast-like cells in the human salivary cancer cells occurs in the presence of 22-oxa-1alpha, 25-dihydroxyvitamin D3 and beta-glycerophosphate.

Recurrent intraoral herpes simplex virus infection

A 48-year-old female had primary herpetic gingivostomatitis, followed by recurrent intraoral herpes simplex virus (HSV) disease; HSV isolates were obtained from the swabs of primary and recurrent lesions; restriction endonuclease cleavage analysis of the viral DNAs extracted from Vero cells infected with the HSV isolates according to the method of Hirt was carried out. The viral DNAs were cleaved by restriction endonucleases such as BamHI, KpnI and SalI and resolved by agarose gel electrophoresis, followed by staining with ethidium bromide. Consequently, their cleavage patterns were very similar to one another and were identified as HSV type 1. From these findings, it can be concluded that primary and recurrent lesions of this case are caused by the same virus.