Molecular mechanism of decitabine -induced degradation of DNA methyltransferase 1 (DNMT1) in cancer cells

1594 Decitabine (5-dAzaC) alone or in combination with inhibitors of histone deacetylases is used as a chemotherapeutic agent against leukemia. It is a well-known irreversible inhibitor of DNA methyltransferases. Once incorporated into the DNA it forms a covalent complex with a serine residue at the active site of DNA methyltransferase resulting in its inactivation. Recently we have shown that treatment of mouse lymphosarcoma cells with 5-dAzaC resulted in the decline of Dnmt1 steady state level (Ghoshal et al. Mol. Cell. Biol. 2002, 22:8302-8319). Later on, studies with different types of cancer cells of human, mouse and rat origin have demonstrated that treatment of cells with decitabine not only inactivates DNMT1 but also facilitates its rapid degradation. This nucleoside analog-induced degradation of DNMT1 occurs in a time and concentration dependent manner. Among different protease inhibitors tested, only MG132 and lactacystin blocked 5-dAzaC-induced degradation of DNMT1, implicating its degradation by the proteasomal pathway. To further confirm this phenomenon, we used a cell line (ts20), a Balbc 3T3-derived cell line harboring a temperature-sensitive mutant of ubiquitin-activating enzyme (E1), the first enzyme of proteasomal pathway. E1 in these cells is active when grown at 31°C but is inactivated when growth temperature is shifted at 39°C. DNMT1 was degraded in a time-dependent manner when cells were treated with decitabine at 31°C. In contrast, the inhibitor-induced degradation was significantly impeded in cells preincubated for 24 hr at 39°C. These results further reinforce the notion that the nucleoside analog facilitates ubiquitination and proteasomal degradation of DNMT1. Analysis of the primary structure of DNMT1 revealed existence of a conserved KEN box, a targeting signal for ubiquitination and subsequent proteasomal degradation. Currently we are exploring the role of KEN box, if any, in 5-AzaC-mediated degradation of DNMT1. (supported, in part, by grants, ES 10874, CA 81024)