THE DEVELOPMENT OF A GENE-BASED DIAGNOSTIC METHOD TO EVALUATE THE EFFICACY OF PRE-CHEMOTHERAPUTIC AGENTS IN ORAL CANCER THAT ALTER GENE EXPRESSION BY AFFECTING DNA METHYLATION

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(1)In oral cancer cell (OCC) lines, DNA methylation of the CHFR gene was examined as a target to evaluate the sensitivity of pre-chemotherapeutic agents.(2)To examine the role of CHFR in the prophase mitotic checkpoint in more detail, we used cells which do not express CHFR to evaluate the mitotic index after treatment of microtubule inhibitors such as docetaxel. There is a significant correlation between the absence of CHFR expression and a high mitotic index in OCC lines. Re-expression of CHFR, by blocking promoter methylation through drug treatment (i.e. 5-aza-dC) reduced the mitotic index in cells. This indicates that CHFR regulates a checkpoint importance in controlling entry into mitosis. Thus, cells lacking CHFR apparently do not stop at prophase and enter mitosis when treated with microtubule inhibitors.(3)In addition to oral cancer, abnormality of CHFR was observed in colon cancer, stomach cancer, and leukemia. To verify that the sensitivity of CHFR-deficient OCC to docetaxel is specific to microtubule inhibitors, cells were treated will another microtubule inhibitor (paclitaxel), a topoisomerase inhibitor (VP16), or an alkylating agent (CDDP). Some cells were sensitive to paclitaxel but not to VP16 or CDDP, indicating that the CHFR checkpoint is involved in the response to mitotic stress but not in the response to topoisomerase inhibition or double strand breaks.(4)Disruption of CHFR by short hairpin RNA reduced the ability of cells to regulate G2/M phase transition due to impaired mitotic checkpoint. In addition, CHFR knockdown in OCC increases sensitivity of the cells to microtubule inhibitors. Our results indicate that CHFR can be a molecular target for chemotherapy.