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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 in- he DNA-PK complex is one of the major pathways by which cells respond to DNA double-strand breaks (DSBs). The DNA-PK complex consists of a heterodimer comprising 70-and 80-kDa proteins termed Ku and a 465-kDa serine/ threonine protein kinase catalytic subunit termed DNA-PKcs. 1)The Ku (p70/p80) component functions as an activator of the catalytic subunit, and also represents the major double-stranded DNA binding protein.1, 2) DNA-PK plays an important role in the repair of DSBs and in V(D)J recombination.3) Tumor cell lines defective in the expression of either Ku or DNA-PKcs exhibit marked radiation sensitivity. Cells lacking DNA-PK activity because of defects in DNA-PK components, such as human malignant glioma M059J cells and cells derived from scid mice, show hypersensitivity to ionizing radiation.3-7) These previous laboratory findings suggested that DNA-PK is a candidate as a predictor of cellular radiation sensitivity. There is, however, little information on the expression of DNA-PK in primary human tumors and the correlation, if any, with radiation sensitivity, though the results are not definitive. [8][9][10][11] Therefore, the aim of this study is to evaluate the relationship between expression levels of DNA-PK complex proteins and radiation sensitivity. Materials and MethodsCell culture. All SCC cell lines were grown in Ham/F12: DMEM (1:1) supplemented with 10% fetal bovine serum, 24 µg/ml adenine, 0.4 µg/ml hydrocortisone and 50 units/ml penicillin and streptomycin. The HSC2, HSC3 and HSC 4 cell lines were provided by Japanese Collection Research Bioresources. The SCC15, SCC25, SCC66 and SCC111 cell lines were provided by Dr.

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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 G 1 and G 2 -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 DNAPKc 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 mm 3 in HSC2 cells (p < 0.01) and from 284.2 to 12.4 mm 3 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.