Expression of dUTPase in malignant cells demonstrates significant variation, both in total expression and subcellular localization which may account for differences in clinical response to the TS-inhibiting class of chemotherapeutics (10
). Therefore, elucidating the molecular basis for variable dUTPase expression is of interest from both a basic science and clinical perspective. Using promoter analysis in HCT116 p53+/+
colon cancer cells, the region containing the E2F and triple Sp1 site was found to be sufficient for driving DUT-N promoter activity (A and B). Site-directed mutagenesis of the E2F and Sp1 sites clearly demonstrated that both transcription factors positively contribute to basal promoter activity (C and D). We also report that the dUTPase promoter is highly responsive to Sp1 overexpression in HCT116 colon cells and silent in the absence of Sp1 and responsive to Sp1 expression in Sp1-deficient Drosophila
SL2 cells (A). Furthermore, knockdown of Sp1 resulted in the downregulation of dUTPase protein expression and significantly attenuated dUTPase promoter activity (C). Finally we confirmed the presence of both Sp1 and E2F-1 transcription factors at the proximal dUTPase promoter in vivo
using ChIP and qPCR (D). Interestingly, dysregulation of both Sp1 (33
) and E2F-1 (44–47
) are widely observed following neoplastic transformation and are reported to contribute to a variety of tumor processes including progression, drug resistance, angiogenesis and metastasis. It is therefore likely that dysregulation of E2F-1 and Sp1 expression may contribute to the wide variation in dUTPase expression levels observed in tumor tissues (10
Initial reports identified dUTPase mRNA as modulated by both mutant and wild-type p53 (22
). However, the precise mechanism by which wild-type p53 exerts its repressive effects on dUTPase has not been described. To our knowledge, this report provides the first evidence demonstrating that dUTPase is directly modulated at the transcriptional level by an in vivo
promoter association with p53. Introduction of p53WT
resulted in significant repression of the dUTPase promoter (C and E). Interestingly, the level of repression in the HCT116 p53–/–
cells was significantly greater following introduction of p53WT
when compared to the HCT116 p53+/+
cells, suggesting that expression of p53WT
already exerts a regulatory influence on the dUTPase promoter in the HCT116 p53+/+
cells. This is further supported by the observation that when the p53MUT
is introduced into the p53+/+
cells, the promoter activity is induced to a much greater extent than the p53–/–
cells. This phenomenon may be exacerbated due to the artificial nature of the luciferase system, but would indicate the disruption of an unknown mediator involved in dUTPase promoter regulation by the dysregulated interactions of p53MUT
. This observation has previously been noted in the TSP50 gene which is negatively regulated by p53, where the authors reported that introduction of mutant p53 significantly increased the TSP50 promoter activity in the three cell lines containing p53WT
). These data would further suggest that tumoral p53 status and mutation may be important regulators of dUTPase expression and thus may influence chemotherapeutic response.
It is important to consider that ectopic p53 expression is unlikely to accurately simulate p53 activation in response to DNA damage which involves complex post-translation modifications. We therefore utilized chemotherapeutic agents used in the treatment of colorectal cancer which are known to activate p53 including the DNA-damaging agent oxaliplatin and the TS-inhibitors 5-FU and FUdR. Both 5-FU and FUdR resulted in a modest downregulation of dUTPase protein consistent with previous reports (21
) (A). However, for the first time, we identified oxaliplatin as a potent downregulator of dUTPase at the promoter, mRNA and protein levels (B and 5A). Treatment of the HCT116 p53–/–
cells with the same clinically relevant doses of oxaliplatin failed to modulate the dUTPase promoter or downregulate mRNA and protein expression, indicating a direct role for p53 or a p53-target in the repression (B, 5A and B). Moreover, our findings indicate that this acute downregulation preceded complete cell cycle arrest with rapid repression of the dUTPase promoter and downregulation of the mRNA within 24 h. Although both 5-FU and FUdR result in similar levels of p53 induction, the cytotoxicity induced by these agents is of a significantly different nature than oxaliplatin. Specifically, 5-FU and FUdR are reported to initiate rapid S-phase arrest which is associated with thymidylate depletion as a result of TS-inhibition and is independent of p53 (50
). The induction of p53 in response to 5-FU is reported to be a later event, possibly due to aberrant detection of uracil and fluoronucleotides in synthesized DNA. Oxaliplatin binds with high affinity to GC-rich DNA forming bulky platinum adducts which block DNA replication, activate p53 and stimulate the nucleotide excision repair pathway (51
). Importantly, oxaliplatin treatment in HCT116 cells was previously reported to initiate a p53-dependent cell cycle arrest, indicating an immediate role for p53 in mediating the cellular response to oxaliplatin in the HCT116 colon cancer model (52
). A recent report directly compared the p53 response in colon cancer cells treated with 5-FU compared to the direct DNA-damaging agent doxorubicin. The authors noted that 5-FU treatment did not accumulate p53 or result in p53 phosphorylation until 24 h post-treatment. However, doxorubicin treatment resulted in rapid p53 induction through protein stabilization and noted that p53 protein was both rapidly phosphorylated and acetylated within 4 h of treatment (53
). Furthermore, p53 signaling is a complex pathway involving multiple post-translational modifications which are specific to its subsequent functions which vary markedly from cell-cycle arrest and DNA repair to apoptosis (54
). Considering that oxaliplatin is also a direct DNA-damaging agent, it is possible that the mechanism of resultant p53 activation and associated post-translational modifications are markedly different than those observed following treatment with 5-FU and FUdR and this may account for the difference in the ability of these agents to repress dUTPase gene expression. We also note that ectopic dUTPase protein expression from an exogenous CMV-driven promoter was unaffected by oxaliplatin, discounting protein stability or atypical turnover and supporting a direct transcriptional effect (D). Simultaneous analysis of TS, another classical S-phase gene revealed only a moderate downregulation following oxaliplatin treatment independent of p53 (C), supporting the idea that the presence of a p53 responsive element is a key difference between the regulation of TS and dUTPase.
Previous reports suggested that p53-mediated transactivation of p21 was sufficient and necessary to exert the negative gene regulation of p53 (40
). By using a p21 HCT116 null isogenic cell line, we demonstrate that p21 is not required for the transcriptional repression of dUTPase in response to oxaliplatin, further suggesting a direct role for p53 (). We subsequently confirmed the presence and enrichment of p53 at the dUTPase proximal promoter following treatment with oxaliplatin. Importantly, this was accompanied by reductions in Sp1 and Ac-H3 (A). Although the exact mechanism by which p53 represses the dUTPase promoter is not clear, these observations suggest that p53 interacts with the dUTPase promoter transcriptional machinery in the absence of a p53-specific cis
element to suppress gene expression. In support of our observations, additional reports demonstrated p53-mediated transcriptional repression of the protective antioxidant enzyme manganese superoxide dismutase and the anti-apoptotic molecule MCL-1 in the absence of a p53 consensus site and demonstrated this to be mediated through disruption of normal transcription factor binding (56
). Furthermore, the DNA repair enzyme AP-endonuclease is repressed by p53, in the absence of a cis
element through interfering with Sp1 binding (58
). This model is supported by our findings which demonstrated increased p53 and reduced Sp1 at the dUTPase promoter following oxaliplatin treatment. Interestingly, treatment with mithramycin A, which directly reduced Sp1 enrichment, did not show enrichment for p53, suggesting that displacing Sp1 or additional Sp-family members at the dUTPase promoter or inhibiting additional Sp1-driven gene expression can abrogate the promoter enrichment of p53. In support of this, our observation that the triple Sp1 site-mutant construct, which importantly still retained 50% transcriptional activity of the full-length construct, was not repressed by oxaliplatin and would further indicate that p53 is exerting its repressive effects through the region of the promoter known to bind Sp1 (B). Previous studies have demonstrated that p53 can directly associate with transcription factors in vivo
including Sp1 (57
) and with histone deacetylase 1 (60
) and repress transcription from specific promoters. Importantly, it has also been demonstrated that mutant p53 lacks the capacity to interact with HDAC1 and repress transcription (60
), representing another possible mechanism for the p53-mutant induction of dUTPase previously reported (24
) and our observations in this report showing p53-mutant induction of the dUTPase promoter. Furthermore, the inability of mutant forms of p53 to repress gene transcription may contribute to associated chemoresistance by abrogating apoptosis induced through repression of protective genes.
As a single agent, oxaliplatin has limited clinical activity in colorectal cancer and is primarily used in combination with 5-FU or capecitabine resulting in synergistic anti-tumor activity (3
). Attempts to define the molecular basis for this synergistic interaction have demonstrated that oxaliplatin induces modest suppression of both TS mRNA and activity of the 5-FU catabolic enzyme dihydropyrimidine dehydrogenase (DPD) (62
). In the present study, we confirmed that oxaliplatin induced a modest downregulation of TS mRNA independent of p53 and p21. In addition, we provide the first evidence of a p53-dependent acute downregulation of dUTPase in the HCT116 p53+/+
colon cell line following treatment with a clinically relevant dose of oxaliplatin. When oxaliplatin was combined with a TS-inhibitor, there was a significant increase in the accumulation of dUTP consistent with downregulation of dUTPase and increased effects of TS-inhibition (E). Targeted use of oxaliplatin in tumors with elevated levels of dUTPase and wild-type p53 may represent a novel strategy for improving the therapeutic efficacy of TS-directed agents and warrants further investigation ().
Figure 8. Working model of p53-mediated repression of dUTPase transcription and subsequent enhancement of 5-FU-mediated downstream effects. Schematic diagram illustrating the downstream effects of p53-mediated transcriptional repression of dUTPase following treatment (more ...)
Additional genes with critical roles in cytoprotection and survival including anti-apoptotic, oxidative stress elimination and DNA repair genes have previously been demonstrated to be repressed by p53, promoting the hypothesis that the pro-apoptotic function of p53 is mediated in part through transcriptional repression of cytoprotective genes. It is therefore plausible that the p53-mediated repression of dUTPase may enhance DNA damage and represent one component in the commitment to irreversible cell death and contribute to the tumor suppressing functions governed by p53.
In conclusion, p53 plays a critical role in the transformation and progression of many human malignancies, thus, the effect of p53 on uracil–DNA metabolism may increase our understanding of the complex regulatory pathways governed by p53 and may assist in providing more targeted chemotherapy. Our results have identified and characterized a novel mechanism of p53-dependent transcriptional regulation of an evolutionary conserved and essential enzyme involved in regulating intracellular uracil pools and maintaining genomic stability.