Chromosome 1 open reading frame 10 (C1orf10) gene is frequently down-regulated and inhibits cell proliferation in oral squamous cell carcinoma.

Chromosome 1 open reading frame 10 (C1orf10) is a recently identified gene encoding a protein with an S100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected Clorf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G1 phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle.

Status of reduced expression and hypermethylation of the APC tumor suppressor gene in human oral squamous cell carcinoma.

The adenomatous polyposis coli gene (APC gene) originally was identified as a tumor suppressor gene in colon cancer. We reported previously that APC is mutated and/or deleted in primary oral squamous cell carcinoma (OSCC) tissues and suggested that loss of APC function contributes to carcinogenesis in the oral region. In this study, we examined 50 OSCC tissue samples, which had been fixed in 10% buffered formaldehyde solution and embedded in paraffin, and eight cell lines, which were derived from OSCC, to analyze the expression level of the APC gene. Significant down-regulation of APC was detected by immunohistochemistry in 15 (30.0%) of 50 tissue samples and by the reverse transcriptase-polymerase chain reaction in five (62.5%) of eight cell lines. We then investigated the status of APC gene promoter methylation and restoration of the APC gene mRNA. Hypermethylation of the APC promoter CpG island was detected in two of eight (25%) OSCC-derived cell lines, and APC gene mRNA was restored in all OSCC-derived cell lines showing down-regulation of gene expression (n=5) after treatment with 5-aza-2'-deoxycytidine, a DNA demethylating agent. Thus, the contribution of down-regulated APC expression to the development of human OSCC was about 30%, and hypermethylation of the gene promoter CpG island was confirmed to be a significant mechanism of inactivation of the APC gene in oral carcinogenesis.

Down-regulation of DMBT1 gene expression in human oral squamous cell carcinoma.

Deleted in malignant brain tumors 1 (DMBT1) gene was recently isolated on chromosome 10q25.3-26.1 and has been proposed as a putative candidate tumor suppressor for brain, esophageal, gastric, colorectal, and lung cancer. However, little is known about the association of DMBT1 with oral squamous cell carcinoma (OSCC). To study the role of DMBT1 gene in OSCC oncogenesis, we examined 9 OSCC derived cell lines and 45 primary OSCC tissue specimens with respective normal tissues. Semi-quantitative reverse transcriptase chain reaction (RT-PCR) analysis revealed down-regulation or deletion of DMBT1 expression in all of the 9 cell lines and in 18 (40%) of 45 primary OSCC tissues. Additionally, 57 OSCC tissue specimens were examined by immunohistochemical staining of protein showing down-regulation of DMBT1 protein in 31 (56.1%) of the 57 primary OSCC tissue specimens. To assess restoration of DMBT1 expression by demethylation of promoter region, the 9 cell lines were treated with 5-aza-2-deoxycytidine (5-Aza-C), one of the DNA demethylating agents. Six (66.7%) of 9 cell lines demonstrated restoration of DMBT1 expression after 5-Aza-C treatment. These results suggest that DMBT1 gene is involved in OSCC oncogenesis and/or progression and that methylation of promoter region is one of the important mechanisms suppressing the DMBT1 gene expression.

In-house cDNA microarray analysis of gene expression profiles involved in SCC cell lines.

To analyze gene expression in oral cancer, we produced a specialized in-house cDNA microarray. The cDNA library was constructed from surgical specimens of oral squamous cell carcinoma (SCC) using an oligo-capping method. cDNA clones (n=4,800) were randomly selected and their 5'-end nucleotide sequences were determined. Overlapping clones were excluded, and 1,423 independent clones were selected and used for microarray production. Compared to the public nucleotide sequence database, 61% of our cDNA clones were full-length. By correlating expression patterns across SCC cell lines, we identified 53 genes (7 up-regulated and 46 down-regulated) that are differentially expressed in SCC cell lines compared to normal mucosa. Semi-quantitative RT-PCR analysis confirmed these findings and validity of our cDNA microarray. Using specimens from SCC patients, we investigated the expression status of the IL-1ra gene, which showed the down-regulated gene by microarray analysis. Gene expression clearly fell in the SCC specimens relative to their references, which indicated that our in-house cDNA microarray system rapidly identified and characterized candidate biomarkers for clinical use.

Enhanced expression of the LDH-A gene after gravity-changing stress in human RSa cells.

A major issue in radiation and space biology is whether gene expression levels are altered in cells exposed to gravity-changing stress. In the present study, genes up- or down-regulated in radiation-sensitive human RSa cells cultured under gravity-changing conditions, were identified using a PCR-based mRNA differential display method. Exposure of cells to gravity-changing stress was performed by free-fall with a drop-shaft facility or by an airplane-conducted parabolic flight. Among the candidates for gravity-changing stress-responsive genes obtained by the differential display analysis, the lactate dehydrogenase A gene (LDH-A) was confirmed by Northern blotting analysis to exhibit increased expression levels. The gravity-changing stress consisted of a combination of microgravity and hypergravity. However, exposure of the cells to hypergravity produced by centrifuge only slightly affected the LDH-A mRNA expression. Thus, LDH-A was found to be a candidate for the genes which play a role in the cellular response to gravity-changing stress, and mainly to microgravity.