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It is known that the O 6 -methylguanine-DNA methyltransferase (MGMT) gene is susceptible to epigenetic regulation associated with an altered frequency of CpG methylation. To investigate whether epigenetic regulation of the MGMT gene might lead to significant reductions in the expression levels of cancer cells, we sought evidence of a link between the methylation status of the MGMT promoter and the expression levels of seven human oral cancer cell lines. We found two frequently methylated regions: the 5' region extending from nt 690 to nt 893 in the promoter, and the more 3' region extending from nt 1060 to nt 1151 in the untranslated first exon. The 3' region was hypermethylated independently of MGMT expression levels in all cell lines. By contrast, in the three MGMT-downregulated cell lines (SAS, Hep2, HO-1-u-1), the levels of MGMT expression were inversely related to the density of 5' region of the methylated CpGs in the MGMT promoter. Our results implied that the transcriptional inactivation of MGMT might require methylation of the 5' region, but not that of the 3' region in oral cancer cell lines. We further explored the role of methylation in MGMT expression by treating cells with 5-Aza-2'-deoxycytidine (5Aza-dC). 5Aza-dC treatment led to the partial or complete cytosine demethylation of two frequently methylated MGMT regions in all cell lines. 5Aza-dC succeeded in upregulating of the MGMT mRNA levels in only 2 of 7 cell lines (HSC3 and HO-1-u-1), and in fact reduced MGMT mRNA in the other 5 cell lines. Furthermore, 5Aza-dC had an inhibitory effect on MGMT protein levels in all cell lines. Our results suggest that MGMT levels may not revert after 5Aza-dC treatment. Based on our findings, the regulation of MGMT expression appears to be more complex than previously thought, although it is at least partially influenced by CpG methylation. Accordingly, care should be taken interpreting the link between MGMT methylation and expression.

We report herein the effects of p53 gene therapy in the radiotherapy or thermotherapy of eight human head and neck squamous cell carcinoma (SCC) cell lines. The discrepancy between radiosensitivity combined with p53 gene therapy than that without p53 gene therapy increased among the eight SCC cell lines. The discrepancy increased in the thermosensitivity at 43 degrees C and decreased in that at 44 degrees C among the eight SCC cell lines. Thus, the p53 gene therapy did not always improve the discrepancy between radiosensitivity and thermosensitivity in the eight SCC cell lines. In the radiotherapy combined with adenoviral p53 gene therapy, the survival rates of three of eight SCC cell lines decreased, and that of only one cell line increased compared with radiotherapy alone. In thermotherapy combined with p53 gene therapy, the survival rates of three at 44 degrees C and five at 43 degrees C of the eight SCC cell lines decreased, although only one cell line at 43 degrees C increased its survival rate compared with thermotherapy alone. The p53 gene therapy decreased the survival rates of both radiotherapy and thermotherapy in three of eight SCC cell lines. Further, the distribution of plots on the basis of the time for 10% survival of radiotherapy and the dose for 10% survival of thermotherapy with p53 gene therapy shifted to the lower left side of the plots compared with those without p53 gene therapy. These findings indicated that p53 gene therapy improves the effects of both radiotherapy and thermotherapy.

We examined effects of recombinant p53-expressing adenovirus combined with thermoradiotherapy in 8 head and neck squamous cell carcinoma (SCC) cell lines to improve the outcomes of the treatment of advanced head and neck cancer. The p53 gene therapy did not improve the discrepancy between thermoradiosensitivities among the 8 SCC cell lines. However, p53 gene therapy improved the effects of thermoradiotherapy in all 8 cell lines, and there were significant differences in four situations of the HSC4 44 degrees C (p=0.032), SAS at 44 degrees C (p=0.029), the KB at 43 degrees C (p=0.025), and the Ca9-22 43 degrees C (p=0.020). In comparing the survival rates of thermoradiotherapy with those of thermotherapy and radiotherapy, thermoradiotherapy demonstrated actual survival rates less than theoretical survival rates based on the survival rates of thermotherapy multiplied by the survival rates of radiotherapy in almost all treatments of thermoradio-gene therapy of the 8 SCC cell lines. These results demonstrate that thermoradiotherapy combined with p53 gene therapy may be a useful tool in treating SCC cells.

We reconstructed the recombinant p53-expressing adenovirus and examined its infections and effects in head and neck squamous cell carcinoma cell lines. Eight human head and neck squamous cell carcinoma cell lines were infected by the recombinant adenovirus harboring the lacZ gene (AxCAiLacZ) or the wild-type p53 gene (AxCAip53), and the effects were investigated. The eight cell lines were successfully infected by AxCAiLacZ at a level of more than 50%. The survival of all 8 squamous cell lines were inhibited in the range from 8 to 26.7% by only one treatment of the AxCAip53 infection. This result suggested that p53 gene therapy might become a useful tool in head and neck squamous cell carcinoma treatment.