Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and nonamplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus.

DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3' from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5' from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.

[The Molecular Aspect of the Antitumor Effect of Oxaliplatin in Combination with 5-FU].

Platinum-based chemotherapeutic drugs as a component of combination chemotherapy are widely used in the treatment of cancer. In particular, oxaliplatin(L-OHP), one such platinum-based chemotherapeutic drug, has a synergistic effect in combination with 5-FU and Leucovorin for the treatment of advanced colorectal cancer. However, the underlying molecular mechanism of this synergistic effect has not been fully clarified yet. In this review, we summarize several updates about the in vitro action of oxaliplatin in human tumor cells and discuss the underlying mechanism of its synergistic effect with 5-FU.

Nucleolar protein PES1 is a marker of neuroblastoma outcome and is associated with neuroblastoma differentiation.

Neuroblastoma (NB) is a childhood malignant tumor that arises from precursor cells of the sympathetic nervous system. Spontaneous regression is a phenomenon unique to NBs and is caused by differentiation of tumor cells. PES1 is a multifunctional protein with roles in both neural development and ribosome biogenesis. Various kinds of models have revealed the significance of PES1 in neurodevelopment. However, the roles of PES1 in NB tumorigenesis and differentiation have remained unknown. Here we show that NB cases with MYCN amplification and clinically unfavorable stage (INSS stage 4) express higher levels of PES1. High PES1 expression was associated with worse overall and relapse-free survival. In NB cell lines, PES1 knockdown suppressed tumor cell growth and induced apoptosis. This growth inhibition was associated with the expression of NB differentiation markers. However, when the differentiation of NB cell lines was induced by the use of all-trans retinoic acid, there was a corresponding decrease in PES1 expression. Pes1 expression of tumorspheres originated from MYCN transgenic mice also diminished after the induction of differentiation with growth factors. We also reanalyzed the distribution of PES1 in the nucleolus. PES1 was localized in the dense fibrillar component, but not in the granular component of nucleoli. After treatment with the DNA-damaging agent camptothecin, this distribution was dramatically changed to diffuse nucleoplasmic. These data suggest that PES1 is a marker of NB outcome, that it regulates NB cell proliferation, and is associated with NB differentiation.

Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex.

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the three-dimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNA-interacting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.

CtIP- and ATR-dependent FANCJ phosphorylation in response to DNA strand breaks mediated by DNA replication.

FANCJ, also called BACH1/BRIP1, is a 5'-3' DEAH helicase, whose mutations are known as a risk factor for Fanconi anemia and also breast and ovarian cancer. FANCJ is thought to contribute to DNA double-strand break (DSB) repair and S-phase checkpoint through binding to multiple partner proteins, such as BRCA1 and TopBP1, but its molecular regulation remains unclear. We focused on DNA damage-induced phosphorylation of FANCJ and found that reagents that cause DSB or replication fork stalling induce FANCJ hyperphosphorylation. In particular, camptothecin (CPT) induced rapid and efficient FANCJ hyperphosphorylation that was largely dependent on TopBP1 and ATM-Rad3 related (ATR) kinase. Furthermore, DNA end resection that exposes single-strand DNA at the DSB site was required for hyperphosphorylation. Interestingly, upon CPT treatment, a dramatic increase in the FANCJ-TopBP1 complex was observed, and this increase was not alleviated even when ATR-dependent hyperphosphorylation was suppressed. These results suggest that FANCJ function may be modulated by hyperphosphorylation in a DNA end resection- and ATR-dependent manner and by FANCJ-TopBP1 complex formation in response to replication-coupled DSBs.

Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex.

Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with the cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction, with unprecedented configurations of the two molecules. Electron microscopic analyses indicated that this complex structure exists in solution. Our structural study revealed that an interaction occurs between a stretched loop of PCNA and the PfuPol Thumb domain, in addition to the authentic PCNA-polymerase recognition site (PIP box). Comparisons of the present structure with the previously reported structures of polymerases complexed with DNA, suggested that the second interaction plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. This putative mechanism for fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site caused enhancements in the exonuclease activity in the presence of PCNA.

Biochemical properties and base excision repair complex formation of apurinic/apyrimidinic endonuclease from Pyrococcus furiosus.

Apurinic/apyrimidinic (AP) sites are the most frequently found mutagenic lesions in DNA, and they arise mainly from spontaneous base loss or modified base removal by damage-specific DNA glycosylases. AP sites are cleaved by AP endonucleases, and the resultant gaps in the DNA are repaired by DNA polymerase/DNA ligase reactions. We identified the gene product that is responsible for the AP endonuclease activity in the hyperthermophilic euryarchaeon, Pyrococcus furiosus. Furthermore, we detected the physical interaction between P. furiosus AP endonuclease (PfuAPE) and proliferating cell nuclear antigen (PCNA; PfuPCNA) by a pull-down assay and a surface plasmon resonance analysis. Interestingly, the associated 3'-5' exonuclease activity, but not the AP endonuclease activity, of PfuAPE was stimulated by PfuPCNA. Immunoprecipitation experiments using the P. furiosus cell extracts supported the interaction between PfuAPE and PfuPCNA in the cells. This is the first report describing the physical and functional interactions between an archaeal AP endonuclease and PCNA. We also detected the ternary complex of PfuPCNA, PfuAPE and Pfu uracil-DNA glycosylase. This complex probably functions to enhance the repair of uracil-containing DNA in P. furiosus cells.

Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14.

Prevotella bryantii B(1)4 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded beta-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k(cat)/K(m) for pNP-beta-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k(cat)/K(m) for pNP-beta-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between beta-xylosides and beta-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in methanoarchaeon Methanothermobacter thermautotrophicus cells.

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[(14)C]inositol 1-phosphate, which was enzymatically prepared from [(14)C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.

Studies on the base excision repair (BER) complex in Pyrococcus furiosus.

We have been studying the functions of PCNA (proliferating-cell nuclear antigen) for the assembly and reassembly of the replisome during replication fork progression. We have identified the functional interactions between PCNA and several proteins involved in DNA replication and repair from Pyrococcus furiosus. We recently reported that the activity of UDG (uracil-DNA glycosylase) in P. furiosus (PfuUDG) is stimulated by PCNA (PfuPCNA) in vitro, and identified an atypical PCNA-binding site, AKTLF, in the PfuUDG protein. To understand further the function of the complex in the BER (base excision repair) process, we investigated the AP (apurinic/apyrimidinic) endonuclease, which can process the BER pathway after uracil removal by UDG. Interestingly, one candidate ORF (open reading frame) for the AP endonuclease was found in the operon containing the gene encoding UDG in the P. furiosus genome. However, this ORF did not exhibit any activity. Instead, we identified the AP endonuclease activity from the other candidate gene products, and designated the protein as PfuAP. We discovered a physical interaction between PfuAP and PfuPCNA, suggesting the formation of a BER complex in one of the repair systems in P. furiosus.

Direct visualization of DNA baton pass between replication factors bound to PCNA.

In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process.

The closed structure of an archaeal DNA ligase from Pyrococcus furiosus.

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 A resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two "catalytic core" domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the "domain-connecting" role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.

PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma.

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed in vitro sphere formation, derepressed its target genes, and suppressed in situ tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. Cancer Res; 77(19); 5259-71. ©2017 AACR.

Neurocan, an extracellular chondroitin sulfate proteoglycan, stimulates neuroblastoma cells to promote malignant phenotypes.

Neurocan (NCAN), a secreted chondroitin sulfate proteoglycan, is one of the major inhibitory molecules for axon regeneration in nervous injury. However, its role in cancer is not clear. Here we observed that high NCAN expression was closely associated with the unfavorable outcome of neuroblastoma (NB). NCAN was also highly and ubiquitously expressed in the early lesions and terminal tumor of TH-MYCN mice, a NB model. Interestingly, exogenous NCAN (i.e., overexpression, recombinant protein and conditioned medium) transformed adherent NB cells into spheres whose malignancies in vitro (anchorage-independent growth and chemoresistance) and in vivo (xenograft tumor growth) were potentiated. Both chondroitin sulfate sugar chains and NCAN's core protein were essential for the sphere formation. The CSG3 domain was essential in the moiety of NCAN. Our comprehensive microarray analysis and RT-qPCR of mRNA expression suggested that NCAN treatment promoted cell division, and urged cells to undifferentiated state. The knockdown of NCAN in tumor sphere cells cultured from TH-MYCN mice resulted in growth suppression in vitro and in vivo. Our findings suggest that NCAN, which stimulates NB cells to promote malignant phenotypes, is an extracellular molecule providing a growth advantage to cancer cells.

Phosphorylation of EB2 by Aurora B and CDK1 ensures mitotic progression and genome stability.

Temporal regulation of microtubule dynamics is essential for proper progression of mitosis and control of microtubule plus-end tracking proteins by phosphorylation is an essential component of this regulation. Here we show that Aurora B and CDK1 phosphorylate microtubule end-binding protein 2 (EB2) at multiple sites within the amino terminus and a cluster of serine/threonine residues in the linker connecting the calponin homology and end-binding homology domains. EB2 phosphorylation, which is strictly associated with mitotic entry and progression, reduces the binding affinity of EB2 for microtubules. Expression of non-phosphorylatable EB2 induces stable kinetochore microtubule dynamics and delays formation of bipolar metaphase plates in a microtubule binding-dependent manner, and leads to aneuploidy even in unperturbed mitosis. We propose that Aurora B and CDK1 temporally regulate the binding affinity of EB2 for microtubules, thereby ensuring kinetochore microtubule dynamics, proper mitotic progression and genome stability.

Neuroblastoma models for insights into tumorigenesis and new therapies.

INTRODUCTION: Neuroblastoma is the most common extracranial solid tumor in children. It occurs in the adrenosympathetic lineage, which is derived from the neural crest. MYCN amplification is found in about 20% of cases and is the most powerful prognostic factor. Anaplastic lymphoma kinase (ALK) mutation is also found in 7% of sporadic neuroblastomas and 50% of familial neuroblastomas. Although several mutations other than ALK are also found, about 70% of neuroblastomas show no mutations. Another important feature of neuroblastoma is that it sometimes spontaneously regresses. These features collectively suggest that neuroblastoma is caused by aberrations in the normal development processes of the neural crest. AREAS COVERED: This review highlights a number of models of neuroblastoma including genetically engineered mouse models (GEMMs). The main GEMMs described here are: tyrosine hydroxylase (TH)-MYCN, TH-MYCN/Trp53(+/-), TH-MYCN/TH-Cre/Casp8(flox/flox), TH-MYCN/TH-ALK(F1174L) and DBH-iCre/CAG-LSL-Lin28b. EXPERT OPINION: The current mouse models available are very useful for investigating the mechanisms of tumorigenesis and for developing therapeutics. However, many aspects have not yet been addressed. These include immediate early events after tumor initiation, epigenetic changes, spontaneous regression and metastasis. On the other hand, the current models do not perfectly recapitulate features of human neuroblastoma. Therefore, humanized mice and new GEMMs should be also considered for future research.

Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2'-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102.

The 1,2-Diaminocyclohexane Carrier Ligand in Oxaliplatin Induces p53-Dependent Transcriptional Repression of Factors Involved in Thymidylate Biosynthesis.

Platinum-based chemotherapeutic drugs are widely used as components of combination chemotherapy in the treatment of cancer. One such drug, oxaliplatin, exerts a synergistic effect against advanced colorectal cancer in combination with 5-fluorouracil (5-FU) and leucovorin. In the p53-proficient colorectal cancer cell line HCT116, oxaliplatin represses the expression of deoxyuridine triphosphatase (dUTPase), a ubiquitous pyrophosphatase that catalyzes the hydrolysis of dUTP to dUMP and inhibits dUTP-mediated cytotoxicity. However, the underlying mechanism of this activity has not been completely elucidated, and it remains unclear whether factors other than downregulation of dUTPase contribute to the synergistic effect of 5-FU and oxaliplatin. In this study, we found that oxaliplatin and dachplatin, platinum-based drugs containing the 1,2-diaminocyclohexane (DACH) carrier ligand, repressed the expression of nuclear isoform of dUTPase (DUT-N), whereas cisplatin and carboplatin did not. Oxaliplatin induced early p53 accumulation, upregulation of primary miR-34a transcript expression, and subsequent downregulation of E2F3 and E2F1. Nutlin-3a, which activates p53 nongenotoxically, had similar effects. Introduction of miR-34a mimic also repressed E2F1 and DUT-N expression, indicating that this miRNA plays a causative role. In addition to DUT-N, oxaliplatin repressed, in a p53-dependent manner, the expression of genes encoding enzymes involved in thymidylate biosynthesis. Consequently, oxaliplatin significantly decreased the level of dTTP in the dNTP pool in a p53-dependent manner. These data indicate that the DACH carrier ligand in oxaliplatin triggers signaling via the p53-miR-34a-E2F axis, leading to transcriptional regulation that ultimately results in accumulation of dUTP and reduced dTTP biosynthesis, potentially enhancing 5-FU cytotoxicity.