Yuuji Nakahara

Inactivation of the p14ARF, p15INK4B and p16INK4A genes is a frequent event in human oral squamous cell carcinomas

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27KIP1 and p15INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27KIP1 and p15INK4B. These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27KIP1 and p15INK4B.

High frequency of homozygous deletion and methylation of p16INK4A gene in oral squamous cell carcinomas

p16(INK4A) inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR-single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1alpha and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1alpha 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16(INK4A) with immunohistochemistry was detected in 71.9% and matched the alteration of p16(INK4A) gene. These results suggest that p16(INK4A) inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16(INK4A) is a useful method.

Alterations of Rb, p16INK4A and cyclin D1 in the tumorigenesis of oral squamous cell carcinomas

Immunohistochemical analysis of Rb, p16INK4A and cyclin D1 expression was performed on 78 oral squamous cell carcinoma (SCC), 46 leukoplakia, and 20 normal mucosa. Rb and p16INK4A expression were observed in all normal mucosa and most of leukoplakia. Lack of Rb and p16INK4A was observed in 56.4 and 67.9% of SCC, respectively. The overexpression of cyclin D1 was not observed in normal mucosa and was observed in 35.9% of SCC. A strong reciprocal relationship between Rb and p16INK4A expression was observed in oral SCC, and all these SCC cases have at least one of the alterations in the Rb pathway.

p12DOC-1, a growth suppressor, associates with DNA polymerase α/primase

p12DOC‐1 is a growth suppressor identified and isolated from normal keratinocytes. Ectopic expression of p12DOC1 in squamous carcinoma cells led to the reversion of in vitro transformation phenotypes including anchorage independence, doubling time, and morphology. Here we report that p12DOC1 associates with DNA polymerase α/primase (pol‐α: primase) in vitro and in cells. The pol‐α:primase binding domain in p12DOC‐1 is mapped to the amino‐terminal six amino acid (MSYKPN). The biological effect of p12DOC1 on pol‐α:primase was examined using in vitro DNA replication assays. Using the SV40 DNA replication assay, p12DOC‐1 suppresses DNA rep lication, leveling at —50%. Similar results were obtained using the M13 single‐stranded DNA synthesis assay. Analysis of the DNA replication products revealed that p12DOC1 affects the initiation step, not the elongation phase. The p12DOC1 suppression of DNA replication is likely to be mediated either by a direct inhibitory effect on pol‐α:primase or by its effect on cyclin‐dependent kinase 2 (CDK2), a recently identified p12DOC‐1‐associated protein known to stimulate DNA replication by phosphorylating pol‐α:primase. p12DOC1 suppresses CDK2‐mediated phosphorylation of pol‐α:primase. These data support a role of p12DOC1 as a regulator of DNA replication by direct inhibition of pol‐α:primase or by negatively regulating the CDK2‐mediated phosphorylation of pol‐α:primase.—Matsuo, K., Shintani, S., Tsuji, T., Nagata, E., Lerman, M., McBride, J., Nakahara, Y., Ohyama, H., Todd, R., Wong, D. T. W. p12DOC‐1, a growth suppressor, associates with DNA polymerase α/primase. FASEB J. 14, 1318–1324 (2000)

Association of Preoperative Radiation Effect with Tumor Angiogenesis and Vascular Endothelial Growth Factor in Oral Squamous Cell Carcinoma

This study examined the relationship between tumor angiogenesis and the radiation‐induced response, evaluated based on pathological changes, in oral squamous cell carcinoma patients treated with preoperative radiation therapy. Forty‐one cases of squamous cell carcinoma treated with preoperative radiation therapy were investigated. Tumor angiogenesis was assessed by scoring the intratumor microvessel density (IMVD). Expression of vascular endothelial growth factor (VEGF) was also evaluated before and after preoperative radiotherapy. There was no correlation between IMVD in the specimens before therapy and the pathological response to radiation therapy. However, radiation therapy decreased IMVD in the specimens after therapy. A significant association was observed between VEGF expression and resistance to radiation therapy: only 4 of the 21 patients whose tumors exhibited a high level (2+ or 3+) of VEGF staining experienced a major (3+ or 4+) pathological response to radiation therapy. Furthermore, an increasing level of VEGF expression after radiation therapy was observed in non‐effective (0 to 2+) response cases. These results suggest that VEGF expression and the induction of this protein are related to radiosensitivity and could be used to predict the effects of preoperative radiation therapy on oral squamous cell carcinoma.

Apoptosis and p53 are associated with effect of preoperative radiation in oral squamous cell carcinomas.

This study was carried out to elucidate whether apoptosis and p53 can be used to stratify oral cancer patients into groups with a favorable or unfavorable response to preoperative radiation therapy. Thirty-two patients were evaluated. The apoptosis index was 1.7+/-0. 9% in the ineffective cases, and it was significantly lower than effective cases (3.2+/-1.2%). While 14 of 16 effective cases (86.7%) did not express p53, 13 of 16 ineffective cases (81.3%) overexpressed p53. These results suggest that mutated p53 in tumors is associated with a poor response to radiation which may be related to evasion of apoptosis in oral cancer.

Expression of a truncated epidermal growth factor receptor in oral squamous cell carcinomas

Epidermal growth factor receptor (EGFR) frequently overexpresses in cancers, including oral squamous cell carcinomas (OSCC). We previously identified a truncated EGFR (tEGFR) in human oral keratinocytes. In this study, we evaluated the prognostic value of tEGFR in 45 cases of OSCC. tEGFR expression inversely correlated with EGFR expression (r=-0.83, P<0.01), decreased with T-stage progression and lymph-node metastasis (P<0.05). The EGFR/tEGFR ratio correlated with the lymph-node metastasis (P<0.05) and survival outcome (hazard ratio =3.601; P<0.05). These results suggest that tEGFR may play an important roles in oral carcinogenesis and that the EGFR/tEGFR ratio may be a prognostic factor for OSCC.