DNA‐PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA‐PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA‐PK complex formation is one of the major pathways by which mammalian cells respond to DNA double‐strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA‐PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA‐PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA‐PK complex proteins increased after radiation treatment, and the increased values correlated with the tumor radiation resistance. Expression of DNA‐PKcs and Ku70 after irradiation was increased in the surviving cells of OSCC tissues irradiated preoperatively. These results suggest that up‐regulation of DNA‐PK complex protein, especially DNA‐PKcs, after radiation treatment correlates to radiation resistance. DNA‐PKcs might be a molecular target for a novel radiation sensitization therapy of OSCC.

Homozygous deletions (HD) provide an important resource for identifying the location of candidate tumor suppressor genes. To identify the tumor suppressor gene in oral cancer, we employed high-resolution comparative genomic hybridization (CGH)-array analysis. We identified a homozygous loss of FAT (4q35), a new member of the human cadherin superfamily, from genome-wide screening of copy number alterations in one primary oral cancer. This result was evaluated by genomic polymerase chain reaction in 13 oral cancer cell lines and 20 primary oral cancers and Southern blot in the cell lines. We found frequent exonic HD of FAT in the cell lines (3/13, 23%) and in primary oral cancers (16/20, 80%). FAT expression was absent in these cell lines. Homozygous deletion hot spots were observed in exon 1 (9/20, 45%) and exon 4 (7/20, 35%). Moreover, loss of gene expression was identified in other types of squamous cell carcinoma. The methylation status of the FAT CpG island in squamous cell carcinomas correlated negatively with its expression. Our results identify mutations in FAT as an important factor in the development of oral cancer and indicate the importance of FATs function in some squamous cell carcinomas.

Molecular blockade of EGFR with either an EGFR MAb or an EGFR TKI enhances the radiosensitivity of human SCCs. In the present study, we investigated whether treatment with the EGFR TKI gefitinib (Iressa, ZD1839) improves the response to radiotherapy in the OSCC cell lines HSC2 and HSC3. We examined potential mechanisms that may contribute to the enhanced radiation response induced by gefitinib. Growth inhibition was observed in vitro with radiation or gefitinib. A cooperative antiproliferative effect was obtained when cancer cells were treated with radiation followed by gefitinib. Cells treated with a combination of radiation and gefitinib arrested in G₁ and G₂–M phases, with a decrease in the S‐phase population. While radiation alone did not significantly affect MEK1/2 and p38 MAPK autophosphorylation, the combination of gefitinib and radiation completely inhibited the downstream signaling of EGFR. Results from DNA damage repair analysis in cultured OSCC cells demonstrated that gefitinib had a strong inhibitory effect on DNA‐PKc pathways after radiation. Tumor xenograft studies demonstrated that the combination of gefitinib and radiation caused growth inhibition and tumor regression of well‐established OSCC tumors in athymic mice; tumor volume was reduced from 1,008.2 to 231.4 mm³ in HSC2 cells (p < 0.01) and from 284.2 to 12.4 mm³ in HSC3 cells (p < 0.01). Immunohistochemical analysis of OSCC xenografts revealed that gefitinib caused a striking decrease in tumor cell proliferation when combined with radiotherapy. Overall, we conclude that gefitinib enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.