Down-regulated expression of SERPIN genes located on chromosome 18q21 in oral squamous cell carcinomas.

Serpins (serine protease inhibitors) are known as a diverse family of protease inhibitors; however, various other biological activities including tumor suppression, have been recently reported for these molecules. To clarify whether members of the serpin family are involved in OSCC (oral squamous cell carcinoma), global gene screening using microarray analysis was performed with OSCC-derived cell lines. A trend toward diminished expression was shown for some SERPIN genes located on 11q12-q13.1 and 18q21. mRNA expression of SERPIN genes at these chromosome regions was therefore analyzed using real-time quantitative RT-PCR (qRT-PCR) in 55 OSCC samples and matched normal tissue. Statistically significant decreases in expression were found for SERPINB12 (P=0.001), SERPINB13 (P=0.001), SERPINB4 (P=0.042), SERPINB3 (P<0.001), SERPINB11 (P<0.001), SERPINB7 (P=0.021) and SERPINB2 (P=0.018). All of these genes are located on 18q21, the known location of the serpin gene cluster. The results strongly suggest that this chromosome region plays a crucial role in OSCC. Some serpin members in the region might be involved in tumor suppression, or there might be unidentified tumor suppressor genes within or near the chromosome region.

Establishment and characterization of a cisplatin-resistant oral squamous cell carcinoma cell line, H-1R.

Cisplatin (CDDP) is a widely used potent chemotherapeutic agent for many malignancies. However, the mechanism of resistance to CDDP remains unclear. To investigate the molecular mechanism, we established a CDDP-resistant cell line (H-1R) from a CDDP-sensitive cell line (H-1) which was derived from moderately differentiated squamous cell carcinoma of the lower gingiva. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R had a 10-fold greater resistance to CDDP than H-1. When we compared gene expression levels in the cell lines using an in-house cDNA microarray, which represented 2,201 genes originating from normal oral tissue, primary oral cancer, and oral cancer cell lines, 12 genes showing elevated mRNA expression in H-1R compared with H-1 were identified. Among them, the up-regulated expression of ATP-binding cassette transporter genes (MDR1, MRP1, and MRP2), CD55, and PGK1 and down-regulated expression of Caveolin 1 were further confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (PCR) or real-time PCR. Our results suggest that H-1 and H-1R cell lines could be useful for elucidating the candidate genes responsible for CDDP resistance, including the genes found in this study.

Down-regulation of FEZ1/LZTS1 gene with frequent loss of heterozygosity in oral squamous cell carcinomas.

Allelic deletions on the short arm of chromosome 8 (8p) are frequent events in several human malignancies, including oral cancer. We have examined and found two common regions of deletion on 8p (8p12, 8p22) in oral squamous cell carcinomas (SCC)s. The possible involvement of FEZ1/LZTS1 (FEZ1) gene, a candidate tumor suppressor gene, mapped at 8p22, was also evaluated. Here we analyzed whether FEZ1 alterations play a role in the development and progression of oral SCCs. In the present study, we examined FEZ1 expression in 31 primary oral SCCs and 8 SCC-derived cell lines by reverse transcription-PCR (RT-PCR). Thirty-five percent of tumors (11 of 31) and 100% of cell lines (8 of 8) showed absent or reduced mRNA gene expression. To investigate the mechanism for silencing, cells were cultured with 5-aza-2'-deoxycytidine and all the cell lines showed restoration by the demethylating agent. These findings suggest that inactivation of the FEZ1 gene may contribute to the development of oral SCCs.

High prevalence of decreased expression of KAI1 metastasis suppressor in human oral carcinogenesis.

PURPOSE: KAI1 was originally identified in prostate cancer as a metastasis suppressor gene. Recent studies have shown a frequent down-regulation of KAI1 expression in many tumor types, whereas mutation or hypermethylation of the gene is infrequent. The aim of the present study was to examine whether loss of KAI1 expression that might be caused by genetic or epigenetic alterations could contribute to oral carcinogenesis. EXPERIMENTAL DESIGN: We analyzed mutational and methylation status of the KAI1 gene and both the mRNA and protein level in a series of oral tumors [28 precancerous lesions, 101 primary oral squamous cell carcinomas (OSCCs), and 30 metastatic OSCCs] and OSCC-derived cell lines. We also examined p53 protein expression, which has been reported to be a candidate activator for the KAI1 gene. RESULTS: With the exception of three microsatellite instabilities in the KAI1 gene, we found no mutations in the coding sequence of the KAI1 gene, no loss of heterozygosity, and no hypermethylation of the KAI1 promoter region in all samples investigated. By immunohistochemistry, however, high frequencies of KAI1 down-regulation were evident not only in the metastatic OSCCs [29 of 30 (97%)] but also in the primary OSCCs [83 of 101 (82%)] and in the precancerous lesions [13 of 28 (46%)]. There was a significant relationship between down-regulation of KAI1 protein expression and primary tumors associated with lymph node metastases (P = 0.0115), whereas there was no statistical correlation between p53 status and KAI1 expression. Taken together, reverse transcription-PCR data were consistent with the protein expression status in 16 patients from whom mRNA was available. CONCLUSIONS: Our data suggest that whereas loss of KAI1 protein expression is associated with primary tumors with lymph node metastases, the down-regulation of KAI1 is an early event in the progression of human oral cancer. The down-regulation of KAI1 is not associated with either mutation, allelic loss, methylation of the promoter, or p53 regulation.