DNA ligase IV expression was also

DNA ligase IV expression was also enhanced in DNR-resistant subclones (Fig. 1). KU80- and DNA ligase IV-deficient plants are reportedly sensitive to ionizing radiation [53]. The regulatory mechanism of DNA ligase IV has not been reported yet. Sp1 expression reduced by siRNA and Sp1 inhibitor suppressed DNA ligase IV expression (Fig. 6). Online information indicates at least three transcription start sites, but the relative contribution of these transcription start sites has not been elucidated. One promoter type we focused on contained one Sp1 binding site and its promoter analysis revealed the positive involvement of this site on DNA ligase IV transcription (Fig. 8a and Supplementary Fig. 4). Furthermore, in our ChIP assay, increased direct binding of Sp1 to the Sp1 binding site of the 5′ promoter region of the variant 3 DNA ligase IV was shown in K562/DNR Almorexant sale (Fig. 8). However, further analysis is needed to elucidate the DNA ligase IV transcription mechanism in chemo-resistant cells. MDR1 and Bcl-xL overexpressions were also observed in K562/DNR cells (Fig. 1). DNR has been demonstrated to induce MDR1 expression in K562 cells [54]. As shown in Fig. 6b, modulation of Sp1 and Sp3 affects MDR1 but not Bcl-xL expression of K562/DNR cells. The importance of Sp1 in MDR1 transcription has been reported previously [29]. Regulation of the MDR1 promoter has been shown to be dependent on the cyclic AMP-dependent protein kinase and the transcription factor, Sp1 [55]. We also demonstrated higher promoter activity of K562/DNR cells against both MDR1 USP and DSP (Supplementary Fig. 3), supporting the theory that the Sp transcription factor is responsible for MDR1 overexpression of K562/DNR cells. In addition to Sp1, other transcription factors including FOXO3a and STAT5 have been demonstrated to cause MDR1 overexpression of K562 cells [54], [56], [57], suggesting the presence of heterogeneous pathways that induce MDR1 transcription. The involvement of FOXO3 and STAT5 in DNA-PKcs and DNA ligase IV has not been reported before, and the search by the ECR browser (http://ecrbrowser.decode.org) revealed that 1kb of the 5′ promoter of DNA-PKcs possesses 4 STAT (−908bp to −901bp, −676bp to −656bp, −533bp to −526bp and −66bp to −59bp) and no FOXO binding site, whereas the 5′ promoter of DNA ligase IV we analyzed possesses no STAT or FOXO binding sites. The effect of STAT binding site (−66bp to −59bp) was negligible (Fig. 3b), however, our preliminary analysis revealed that other two sites (−676bp to −656bp, and −533bp to −526bp) were responsible for some promoter activity (data not shown). However, the important point is that the difference in the DNA-PKcs promoter activity between K562 and K562/DNR was derived from the region between −49bp and the first exon. The reason for the absence of MDR overexpression in HL60/DNR cells is not known but the absence of an important transcription factor would be suspected. Inhibitors against DNA-PKcs, ATM and MDR1 protein have been reported to overcome fludarabine resistance in CLL cells [57]. Thus, it seems likely that components of NHEJ including DNA-PKcs and DNA ligase IV or their common transcription factor, Sp1, are the medicinally treatable target for overcoming the MDR phenotype. However, further work is needed to rule out unexpected adverse effects by inhibiting housekeeping transcription factors such as Sp1.