PD 169316 Sp family protein has been

Sp family protein has been regarded as the housekeeping gene and is ubiquitously expressed. Although Sp family expression is regulated with some stimuli [17], [20], the total Sp family protein levels are not changed in most cases, as observed in our present study (Fig. 4). Constitutive binding of the low mobility forms of Sp1 and AP-1 has been observed in araC-resistant HL60 PD 169316 [40], and the involvement of Sp1 in response to DNA damage has been reported before [41], [42]. In fact, DNA-PKcs inhibition with NU7026 and siRNA for DNA-PKcs enhanced the sensitivity to DNR in K562/DNR cells (Fig. 2). Moreover, genes actively transcribed by DNA damage such as Gadd45, DDB1/2 and XRCC1 have been shown to possess Sp1 sites in their promoter region [43], [44], [45]. We and others have reported that doxorubicin influences the expression of neutral sphingomyelinase 2 (NSMase2) and glucosylceramide synthase (GCS) through the Sp1 site of respective promoters [17], [46]. Up-regulation of both DNA-PKcs and Sp1 protein of colorectal cancer has been demonstrated only by a clinical immunohistochemical study [47]. Sp1 is a zinc-finger protein constitutively activating housekeeping genes, however, it has been recently reported that Sp1 mediates the inducible gene regulation [48]. Furthermore, Sp1 is phosphorylated in response to DNA damage by Ataxia Teleangiectasia-Mutated kinase [49] or other kinases. Phosphorylation of Sp1 serine 101 residue was reported by UV-irradiation or hydroxyurea treatment [50]. Although it has recently been reported that Sp1 facilitates DSB repair through a non-transcriptional mechanism [51], the regulatory mechanism of DNA-PKcs by activated Sp1 transcription factor was, for the first time, described in the present study. Considering the inhibitory effect of MMA and siRNAs for Sp1 and Sp3 on DNA-PKcs expression, increased Sp1 phosphorylation in K562/DNR was at least partially responsible for the observed increase of DNA-PKcs expression. Similarly, we have previously reported that activated Sp1 but not total Sp1 is responsible for NSMase2 transcription in ATRA-treated MCF-7 cells [52]. 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 cells (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.