Localization and copy number of the protein-coding genes actin, alpha-tubulin, and HSP90 in the nucleus of a primitive dinoflagellate, Oxyrrhis marina.

Chromosomes of the dinoflagellate Oxyrrhis marina are composed of thin parallel filaments running longitudinally and lack the arched structure common to the dinochromosome. The physicochemical and molecular organization of these chromosomes, including the localization and arrangement of genes, is still unknown. We investigated the locations of three protein-coding genes, actin (AF482402), alpha-tubulin (AF482403), and HSP90 (AY391258), on these chromosomes using fluorescence in-situ hybridization (FISH). Primers for these three genes were designated according to known partial sequences. PCR products amplified from total DNA were labeled with digoxigenin (DIG) by random priming and used as probes. After in-situ hybridization, DIG signals were amplified and visualized with anti-DIG-FITC. The number of signals was 3 +/- 1.3 (n = 90) for actin, 4.1 +/- 1.4 (n = 70) for alpha-tubulin, and 5.5 +/- 1.7 (n = 80) for HSP90. This study is the first to locate protein-coding genes in the nucleus of a dinoflagellate, although the chromosomes were greatly damaged during the FISH process. The copy number of each gene per cell was estimated using real time PCR. Resulting copy numbers of actin, alpha-tubulin and HSP90 were, 33.7, 10.4 and 5.4, respectively.

Clinicopathological significance of carbonic anhydrase 9, glucose transporter-1, Ki-67 and p53 expression in oral squamous cell carcinoma.

Carbonic anhydrase 9 (CA9) is a glycoprotein present on the surface of cell membranes. It is expressed in 90% of renal cancer cells, but not in normal kidney tissue. Immunotherapy targeting CA9 is underway, and our group has also conducted a clinical trial using CA9 as a cancer vaccine and confirmed the induction of cytotoxic T lymphocytes, with efficacy in some cases. Expression of CA9 antigen in oral cancer has not been reported in Japan, but our results indicate that immunotherapy targeting CA9 might be possible. We immunohistochemically observed the expression of antigens such as CA9, Ki-67, glucose transporter-1 (GLUT-1) and p53 in 107 subjects with oral squamous cell carcinoma, and examined their correlation with clinicopathological parameters. Immunostaining analysis showed expression of CA9 in 98% of oral cancer subjects, and the survival rate was significantly lower in subjects with CA9 antigen expression in 50% or more cells (P<0.05). Subjects with poorly differentiated, T4 and lymph node metastasis, or Stage IV cancer with high CA9 expression (≥50%) had a worse outcome than those with low CA9 expression. Although GLUT-1 expression was observed in 98% of subjects, similarly to CA9 expression, no significant correlation between its expression and the survival rate was seen. However, subjects with lymph node metastasis had significantly higher GLUT-1 expression, demonstrating that GLUT-1 could be an indicator of lymph node metastasis. Ki-67 was expressed in 92% of subjects, but no correlation with outcome was observed. Expression of p53 was noted in 78% of subjects, and it was found that many oral cancers have p53 genetic abnormalities, but no correlation between p53 and outcome was observed. It was confirmed that CA9 antigen is expressed in most oral cancer subjects, suggesting the possibility of immunotherapy targeting CA9 antigen in oral cancer.