DNA Topoisomerase II Poison TAS-103 Transactivates GC-Box-dependent Transcription via Acetylation of Sp1*

Drug-induced modifications of transcription factors play important roles in both apoptosis and survival signaling. The data presented here show that the DNA topoisomerase II poison TAS-103 transactivated the SV40 promoter in a GC-box-dependent manner and induced Sp1 acetylation in cells expressing p300. This activity was not observed in cells lacking p300. TAS-103 treatment also enhanced the p300 content of the nucleus and the interaction of p300 with Sp1. Cellular susceptibility to TAS-103 was correlated with p300 expression but not with topoisomerase II expression. Furthermore, the presence of p300 significantly sensitized cancer cells to TAS-103 but not to cisplatin. Taken together, these findings demonstrate novel genomic responses to anticancer agents that modulate Sp1 acetylation and Sp1-dependent transcription in an apoptotic pathway.

Sp1 was one of the first transcription factors to be identified in mammalian cells. It is a member of the zinc-finger family, which functions by binding the GC-box within the DNA sequences of promoters (1,2). Sp1 is expressed ubiquitously in various mammalian cells and is implicated in the transcription of many genes, particularly housekeeping genes and those that are involved in cell growth and development (3,4). Changes in the cellular content of Sp1, the Sp1/Sp3 ratio, and its GC-box DNA binding activity influence the regulation of Sp1 transcriptional activity (5).
Transcriptional co-activators have an inherently low transcriptional activity. Their only active role in the transcriptional process occurs through their interaction with transcription factors (6). p300 is a transcriptional co-activator that can acetylate a variety of transcription factors and histones (7,8). The histones are localized within transcriptionally active euchromatin, and their interaction with p300 plays a critical role in transcription regulation. A wide range of biological processes such as the cell cycle, differentiation, and tumor growth are regulated by p300 (9). This protein has the potential to activate p53 target genes and might thus act as a suppressor of tumor cell growth (10 -12). Recently, p300 has been shown to function together with Sp1 in GC-box-dependent transcription (13,14).
The DNA topoisomerase (topo) 1 II poison and anticancer agent TAS-103 induces cellular acidosis through changes in the mitochondrial membrane potential (15). We demonstrated previously that the Sp1 DNA binding activity and interaction of Sp1 with TATA-binding protein are enhanced under conditions of low pH. Therefore, cellular pH might also be crucial for the expression of Sp1 target genes (16), including TAS-103-induced cellular acidosis.
In this study, we show that the treatment of cells with TAS-103 leads to the acetylation of Sp1, resulting in a dramatic induction of Sp1-dependent promoters. We also demonstrate that p300 expression sensitizes cancer cells to TAS-103. We propose that p300-dependent acetylation of Sp1 triggered through the DNA-damage signaling pathway and cellular acidosis leads to the enhanced expression of Sp1 target genes in TAS-103-induced apoptosis. This discovery has important implications for current views of the molecular mechanisms by which anticancer agents trigger genome-wide responses. These important responses might occur through the posttranscriptional modification of transcription factors.

EXPERIMENTAL PROCEDURES
Reagents, Cell Culture, and Antibodies-TAS-103 was kindly provided by the Taiho Pharmaceutical Co., Ltd. (Tokyo, Japan). Cisplatin and etoposide were purchased from Sigma, and camptothecin was purchased from Daiichi Seiyaku (Tokyo, Japan).
Human epidermoid cancer KB cells were cultured in Eagle's minimal essential medium, human breast cancer MCF7 cells and human glioblastoma T98G cells were cultured in Dulbecco's modified Eagle's medium, and human colon cancer cell lines (HCT15, CaCo2, LoVo, and SW620) were cultured in RPMI 1640 medium (all from Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal bovine serum and 0.292 mg/ml L-glutamine. All of the cells were cultured at 37°C in a humidified chamber containing 5% CO 2 .
An expression plasmid for FLAG-Sp1 was obtained using FLAGtagged Sp1 cDNA fragments (a gift from R. Tjian, University of Cali- fornia, Berkeley, CA), which were cloned into the pcDNA3 vector (Invitrogen) (17). HA-p300 derived from cytomegalovirus was prepared as described previously (18).
Transient Transfection and Luciferase Assay-Transient transfections were performed as described previously (17) with minor modifications. Cells were seeded into 12-well tissue culture plates at a concentration of 4 ϫ 10 4 cells/well. The next day, the cells were transfected with a luciferase-reporter plasmid using Superfect reagent (Qiagen) for 12 h according to the manufacturer's instructions. The ␤-galactosidase expression plasmid pCH110 (Amersham Biosciences) was co-transfected as an internal control. The cells were then washed and incubated at 37°C for 12 h in fresh medium with or without TAS-103. Subsequently, the cells were lysed with reporter lysis buffer (Promega).
Luciferase activity in the lysed cells was detected using a Picagene kit (Toyoinki, Tokyo, Japan) according to the manufacturer's instructions. The light intensity was measured for 2 s with a luminometer (Luminescencer JNRII AB-2300, ATTO, Tokyo, Japan). The results shown represent at least three independent experiments and were normalized to ␤-galactosidase activity measured using an enzyme assay kit (Promega).
Preparation of Cellular Extracts-Nuclear extracts were prepared using buffer C as described previously (17,19). Extracts of whole cells were produced by lysing cells in buffer X comprising 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10% glycerol. The cellular debris was removed from the extract by centrifugation, and the supernatant was stored at Ϫ70°C. The protein concentrations of nuclear and whole-cell extracts were determined using the Bradford method.
Western Blot Analysis-Nuclear and whole-cell extracts were separated on 7.5% SDS-PAGE gels. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) using a semidry blotter. Western blot analysis was performed with an appro-priate dilution of each antibody followed by visualization using enhanced chemiluminescence.
In Vivo Binding Assay-An in vivo binding assay (transient transfection and co-immunoprecipitation assay) was performed as described previously (18) with minor modifications. MCF7 cells growing in 60-mm plates were co-transfected with HA-and FLAG fusion plasmids along with Superfect reagent according to the manufacturer's instructions. Three hours after transfection, the cells were washed with phosphatebuffered saline and the medium was renewed. After 36 h, the medium was discarded and fresh medium was added with or without the anticancer agents (TAS-103 and cisplatin) as indicated. Whole-cell or nuclear extracts were prepared as described above. These were incubated for 2 h at 4°C with 2 g of anti-M2 antibody and 10 l of protein A/G-agarose (Qiagen). The beads were then washed three times with buffer X. The immunoprecipitated samples were analyzed using 7.5% SDS-PAGE followed by Western blot analysis.
Cytotoxicity Assay-Cells were seeded in 96-well tissue culture plates at a concentration of 5 ϫ 10 3 cells/well. On the following day, drugs were added to the medium at increasing concentrations. After 48 h, the surviving cells were assayed with TetraColar ONE (Seikagaku Corporation, Tokyo, Japan) for 2 h at 37°C according to the protocol provided and absorbance was measured at 450 nm. The IC 50 value was defined as the drug concentration needed to reduce cell growth by 50% relative to the control.

TAS-103 Treatment
Markedly Induced SV40 Promoter Activity in a GC-box-dependent Manner-We reported previously that the cellular level of Sp1 increased severalfold in human epidermoid cancer KB cells after exposure to 4 M TAS-103 (17). We also demonstrated that the SV40 promoter, which contains six GC-boxes, was activated markedly by TAS-103 treatment in a dose-dependent manner (Fig. 1A). The activity of SV40 Luc1 was maximal following 4 M TAS-103 treatment for 12 h. To confirm that this effect was GC-box-dependent, we constructed the reporter plasmid SV40 Luc2, which lacks a GC-box. TAS-103 did not induce SV40 promoter activity from this construct (Fig. 1B).
Co-activator p300 Has Critical Roles in TAS-103-induced SV40 Promoter Activity-We next examined TAS-103-induced SV40 promoter activity in various human cancer cell lines. Both KB cells and human glioblastoma T98G cells induced transactivation of the SV40 promoter ϳ80and 30-fold following TAS-103 treatment, respectively, whereas human colon cancer HCT15 cells did not ( Fig. 2A). However, the Sp1 content of the nucleus induced by TAS-103 treatment was increased only severalfold in these three cell lines (data not shown). , and HCT15 cells were treated with TAS-103 (4 ⌴) for 12 h. 50 g of nuclear extract prepared with buffer C was analyzed using 7.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Western blot analysis was performed with an appropriate dilution of anti-p300 (upper panel). The gel was stained with Coomassie Brilliant Blue (CBB) (lower panel). Three independent experiments were performed, and the results were highly consistent. C, exogenous p300 enhances TAS-103-induced SV40 promoter activity. HCT15 cells lacking p300 were transiently transfected with the SV40 Luc1 reporter plasmid and the p300 expression plasmid. After transfection for 12 h, the cells were incubated for a further 24 h in fresh medium followed by incubation in medium with or without TAS-103 (4 M). Luciferase activity was measured as described under "Experimental Procedures." The values shown are the mean Ϯ S.D. of at least three independent experiments. Error bars indicate the mean Ϯ S.D.
Mutations of p300 have frequently been detected in colon cancer cell lines including HCT15 cells (12). p300 expression was induced by treatment with TAS-103 in KB and T98G cells but not in HCT15 cells (Fig. 2B). Furthermore, transient transfection of a p300 expression plasmid was able to enhance TAS-103-induced SV40 promoter activity by ϳ20-fold in HCT15 cells (Fig. 2C). This finding suggests that the induction of Sp1 acetylation might be involved in the molecular mechanisms underlying the TAS-103-induced transactivation of Sp1dependent promoters.
Treatment of Cells with TAS-103 Led to the Acetylation of Sp1 through Interaction with p300 -p300 has been shown to assist Sp1 in GC-box-dependent transcription (13,14). Here, we examined whether the acetylation of Sp1 was led by TAS-103 treatment of the cells. Human breast cancer MCF7 cells, which have greater transfection efficiency than KB cells, were transfected with FLAG-Sp1. The acetylation of immunoprecipitated FLAG-Sp1 occurred at a low level without any stimulation and was significantly induced by TAS-103 treatment but not by cisplatin treatment (Fig. 3A). Suzuki et al. (14) demonstrated the physical and functional interactions between the acetyltransferase region of p300 and the DNA binding domain of the transcription factor Sp1. Therefore, we examined whether this interaction was affected by TAS-103 treatment. Our results showed that TAS-103 treatment significantly enhanced the interaction of Sp1 with p300 (Fig. 3B).
p300 Expression Was Correlated with Sensitivity to TAS-103 Treatment in Human Colon Cancer Cell Lines-Bearing in mind the strong link between p300 expression and Sp1 activation after treatment with TAS-103, we next examined whether p300 was involved in cellular sensitivity to TAS-103. Four human colon cancer cell lines, either with or without p300 expression, were used in a cytotoxicity assay. HCT15 and CaCo2 cells lacked p300 expression, whereas LoVo and SW620 cells showed significant p300 expression (Fig. 4A, upper panel). The levels of topo-II expression varied among these cell lines (Fig. 4A, middle panel). Cellular sensitivity to TAS-103 was dependent upon p300 expression. Cells expressing p300 showed 10 -30-fold greater sensitivity to TAS-103 compared with cells lacking p300 (Fig. 4B). Cellular expression of p300 also sensitized cells to the well characterized topo-II poison, etoposide (Fig. 4C). However, cells expressing p300 showed only a 2-9-fold greater sensitivity to etoposide compared with cells lacking p300. Furthermore, among cells lacking p300, CaCo2 cells showed higher expression of topo-II␣ and higher sensitivity to etoposide than did HCT15 cells. In addition, among the cells expressing p300, LoVo cells showed higher expression of topo-II␣ and higher sensitivity to etoposide than did SW620 cells. These results indicate that topo-II␣ expression might be involved in cellular sensitivity to etoposide when cells are categorized into two groups based on p300 status. On the other hand, the cellular sensitivity to cisplatin was not associated with p300 expression (Fig. 4D). The levels of topo-II expression have been shown to be involved in the sensitivity of cancer cells to topo II poisons (20); however, no such correlation was observed in the sensitivity of these human colon cancer cell lines to TAS-103 (Fig. 4, A and B). These data indicate that the acetylation of Sp1 via an interaction with p300 is involved in TAS-103-induced apoptosis in cancer cells. DISCUSSION We have previously identified several transcription factors involved in genomic responses to anticancer agents including Y-box-binding protein-1 (YB-1), activating transcription factor 4 (ATF4), zinc-finger factor 143 (ZNF143), and mitochon-drial transcription factor A (mtTFA) (21)(22)(23)(24). This study provides evidence to support the novel finding that Sp1 contributes to the stress response of cancer cells following treatment with anticancer agents. We used the SV40 promoter-luciferase reporter to examine whether TAS-103 could activate Sp1-dependent transcription. This was based on the knowledge that Sp1 was originally identified as a transcription factor from HeLa cells that binds to GC-boxes within the 21-bp repeat elements of the SV40 promoter (1, 2). We found that TAS-103 dramatically activated SV40 promoter activity in KB cells (Fig. 1A) by inducing the acetylation of Sp1 through physical interaction with p300 (Fig. 3). Acetylation of Sp1 might be important in bringing about high-level transactivation of the Sp1-dependent promoter. These findings could explain the discrepancy observed in cells expressing p300 such as KB and T98G cells in which TAS-103 induced extensive transactivation of the SV40 promoter ( Fig. 2A), whereas the Sp1 protein level was increased only severalfold by TAS-103 treatment (data not shown). To our knowledge, this study is the first to demonstrate acetylation of Sp1 after treatment with anticancer agents in cancer cells. By contrast, other anticancer agents such as cisplatin, etoposide, and camptothecin did not transactivate the SV40 promoter (data not shown), indicating that TAS-103 might function in these processes through a unique mechanism.
p300 is a transcriptional co-activator and histone acetyltransferase that functions in chromatin remodeling during transcription. Lysine residues that could be acetylated by p300 were clustered within the zinc-finger domain. The acetylated zinc-finger domain of Sp1 might therefore enhance the interaction of Sp1 with GC-boxes to bring about high-level transactivation. It is interesting to note that the nuclear content of p300 was increased in cells treated with TAS-103 (Fig. 2B), indicating that p300 is also involved in drug-induced stress signaling via the acetylation of Sp1.
Drug-induced gene expression is regulated by both transcriptional and posttranscriptional mechanisms. Recently, cellular acidification has been reported in cancer cells after treatment with TAS-103 (15). The cytosolic pH is critical for the cytotoxicity of anticancer agents (25). Cellular acidosis is thought to trigger apoptosis (26,27) and to play an important role in drug resistance (28 -30). We have demonstrated that the GC-box binding activity of Sp1 and its interaction with TATA-binding protein are enhanced under conditions of low pH (16). It is possible that cellular acidosis might stabilize the modification status of Sp1 by regulating the activity of Sp1-modifying enzymes. We have also shown that TAS-103 treatment induces the p300-dependent acetylation of Sp1 (Fig. 3). Of particular interest is the observation that p300 expression significantly sensitizes cancer cells to TAS-103 (Fig. 4, A and B). Unexpectedly, we also found that p300 expression sensitized cells to etoposide, which is another topo-II poison (Fig. 4C). However, cellular sensitivity to etoposide is weaker than the sensitivity to TAS-103. It was shown previously that the antitumor activity of TAS-103 did not correlate with the expression of topo-II in freshly isolated human colon cancer cells, in contrast to etoposide (31). Consistent with this result, cellular sensitivity to TAS-103 did not correlate with the expression of topo-II␣ (Fig.  4, A and B). We also found that etoposide did not enhance SV40 promoter activity, unlike TAS-103 (data not shown). Taken together, these findings indicate that the mode of action of TAS-103 might differ from that of etoposide. Martens et al. (32) reported that p300 is a major constituent of the nuclear matrix, similar to topo-II␣ (33). p300 does not interact directly with topo-II␣. Rather they co-localize with each other. One possible explanation for why p300 expression sensitized cells to etoposide is that both proteins were localized in the nuclear matrixattachment region, which might be critical for inducing a cleavable complex with etoposide.
Our demonstrated results suggest that treatment with TAS-103 might induce apoptosis by activating both p300-and Sp1dependent gene expressions. Sp1 directly or indirectly regulates several proteins including Bax, Fas ligand, and caspase-8 that induce apoptosis (34 -36). Therefore, further studies should examine the relationship between TAS-induced apoptosis and the expression of these genes. Our present data indicate that TAS-103 is potentially unique in its ability to activate Sp1 target genes through both Sp1 acetylation and cellular acidification (Fig. 5). Collectively, these results demonstrate that Sp1 participates directly in the drug-induced signaling pathway through posttranscriptional modification.