Abstract 2560: Cyclin-dependent kinase inhibitor SU9516 enhances sensitivity to methotrexate in human T-cell Jurkat cells

Abstract

Abstract Methotrexate (MTX), a classical anti-folate drug, has been used to treat various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. In fact, it is well known that elevated expression of DHFR is a direct factor in clinical therapeutic resistance to MTX. First of all, we demonstrated that the sensitivity to MTX was enhanced by the knockdown of the DHFR gene in human T-cell leukemia cell. Concretely, the anti-proliferative effect of MTX was significantly enhanced by the knockdown of DHFR expression by siRNA in Jurkat cells. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX. On the other hand, the expression of DHFR is controlled by the transcription factor E2F. Unphosphorylated retinoblastoma (RB) protein is known to bind and inhibit the E2F. Cyclin-dependent kinase (CDK) inhibitors activate RB function and inhibit E2F function. Then we tested whether CDK inhibitor acted as a suppressor of DHFR through the E2F site of the promoter. We found that SU9516, a cyclin-dependent kinase inhibitor, decreased phospho-RB (Ser780) and reduced the expression of both DHFR mRNA and protein, and the DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. Furthermore, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay. To expand these results in Jurkat cells, we have also examined other leukemic cell lines (human T cell leukemia CCRF-CEM cells and human erythroleukemia K562 cells) and confirmed that SU9516 also repressed the levels of DHFR protein and phopho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in both of cell lines. To expand these results by SU9516, we have examined other CDK inhibitors (Purvalanol A and Cdk4/6 Inhibitor IV) and confirmed that these agents also decreased DHFR protein and phospho-RB (Ser780), and enhanced the cytotoxicity of MTX in a colony formation assay in Jurkat cells as similar as SU9516. Based on these findings, we conclude that a combination of cyclin-dependent kinase inhibitors and MTX may be useful for overcoming resistance to MTX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2560.