Idarubicin, an Anthracycline, Induces Oxidative DNA Damage in the Presence of Copper (II).

BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

Functional Analysis of the Role of Equilibrative Nucleobase Transporter 1 (ENBT1/SLC43A3) in Adenine Transport in HepG2 Cells.

Equilibrative nucleobase transporter 1 (ENBT1/SLC43A3) has recently been identified as a purine-selective nucleobase transporter. Although it is highly expressed in the liver, its role in nucleobase transport has not been confirmed yet in hepatocytes or any relevant cell models. We, therefore, examined its role in adenine transport in the HepG2 cell line as a human hepatocyte model. The uptake of [3H]adenine in HepG2 cells was highly saturable, indicating the involvement of carrier-mediated transport. The carrier-mediated transport component, for which the Michaelis constant was estimated to be 0.268 µM, was sensitive to decynium-22, an ENBT1 inhibitor, with the half maximal inhibitory concentration of 2.59 µM, which was comparable to that of 2.30 µM for [3H]adenine uptake by ENBT1 in its transient transfectant human embryonic kidney 293 cells. Although equilibrative nucleoside transporter 1 (ENT1/SLC29A1) and ENT2/SLC29A2 are also known to be able to transport adenine, [3H]adenine uptake in HepG2 cells was not inhibited by the ENT1/2-specific inhibitor of either dipyridamole or nitrobenzylthioinosine. Finally, [3H]adenine uptake was extensively reduced by silencing of ENBT1 by RNA interference in the hepatocyte model. All these results, taken together, suggest the predominant role of ENBT1 in the uptake of adenine in HepG2 cells.

Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter.

Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.

Oxidative DNA Damage and Apoptosis Induced by Aclarubicin, an Anthracycline: Role of Hydrogen Peroxide and Copper.

BACKGROUND/AIM: This study aimed to investigate aclarubicin (ACR)-induced oxidative DNA damage and apoptosis. MATERIALS AND METHODS: ACR-induced apoptosis was analyzed using HL-60 leukemia cells and HP100 cells, hydrogen peroxide (H2O2)-resistant cells derived from HL-60 cells. ACR-induced DNA damage was analyzed using plasmid DNA. RESULTS: HL-60 cells were more sensitive to ACR than HP100 cells. In HP100 cells, DNA ladder formation and caspase-3/7 activity induced by ACR were suppressed or delayed in comparison to those in HL-60 cells. ACR-induced DNA damage occurred in the presence of Cu(II), and scavenger experiments showed that the reactive species causing DNA damage appeared to be generated from H2O2 and Cu(I). Moreover, we detected intracellular Cu(I) induced by ACR in HL-60 cells, using CopperGREEN™, a fluorescent probe for detection of Cu(I) ion specifically. CONCLUSION: ACR-induced DNA damage and apoptosis can be accounted for by the involvement of H2O2 and Cu(I).

Urate transport function of rat sodium-dependent nucleobase transporter 1.

Sodium-dependent nucleobase transporter 1 (SNBT1) is a nucleobase-specific transporter identified in our recent study. In an attempt to search for its potential substrates other than nucleobases in this study, we could successfully find urate, a metabolic derivative of purine nucleobases, as a novel substrate, as indicated by its specific Na+ -dependent and saturable transport, with a Michaelis constant of 433 µmol/L, by rat SNBT1 (rSNBT1) stably expressed in Madin-Darby canine kidney II cells. However, urate uptake was observed only barely in the everted tissue sacs of the rat small intestine, in which rSNBT1 operates for nucleobase uptake. These findings suggested that urate undergoes a futile cycle, in which urate transported into epithelial cells is immediately effluxed back by urate efflux transporters, in the small intestine. In subsequent attempts to examine that possibility, such a futile urate cycle was demonstrated in the human embryonic kidney 293 cell line as a model cell system, where urate uptake induced by transiently introduced rSNBT1 was extensively reduced by the co-introduction of rat breast cancer resistance protein (rBCRP), a urate efflux transporter present in the small intestine. However, urate uptake was not raised in the presence of Ko143, a BCRP inhibitor, in the everted intestinal tissue sacs, suggesting that some other transporter might also be involved in urate efflux. The newly found urate transport function of SNBT1, together with the suggested futile urate cycle in the small intestine, should be of interest for its evolutional and biological implications, although SNBT1 is genetically deficient in humans.

Oxidative DNA Damage Induced by Pirarubicin, an Anthracycline Anticancer Agent, in the Presence of Copper(II).

BACKGROUND/AIM: One mechanism of the anticancer action of anthracyclines is believed to be oxidative DNA damage. Previously, we reported that doxorubicin induced oxidative DNA damage in the presence of Cu(II). However, the mechanism of pirarubicin-induced oxidative DNA damage has not been well clarified. MATERIALS AND METHODS: DNA damage by pirarubicin in the presence of Cu(II) was analyzed using pBR322 plasmid DNA. O2•- derived from pirarubicin in the presence of Cu(II) was detected by cytochrome c reduction. RESULTS: Pirarubicin induced DNA damage in the presence of Cu(II). Scavenger experiments suggest that reactive species are generated from H2O2 and Cu(I). Pirarubicin induced O2•- production in the presence of Cu(II). CONCLUSION: These findings suggest that pirarubicin plus Cu(II) induces oxidative DNA damage in a similar manner to doxorubicin, and Cu(II)-mediated oxidative DNA damage may serve as a common mechanism for antitumor effects of anthracyclines.

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (Km) of 0.23 µM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent Km of 0.36 µM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.

Specific inhibitory effects of myricetin on human proton-coupled folate transporter: Comparison with its effects on rat proton-coupled folate transporter and human riboflavin transporter 3.

Myricetin is a flavonoid that inhibits human proton-coupled folate transporter (hPCFT) in a transient manner, in which inhibition is manifested in its presence, and also in a sustained manner, in which inhibition induced in its presence persists after its removal. In an effort to elucidate the mechanisms involved in those, we examined if myricetin might or might not act similarly on some other transporters. Transporters examined for that, in comparison with hPCFT, were its rat ortholog (rPCFT) and human riboflavin transporter 3 (hRFVT3). Experiments were conducted, using human embryonic kidney 293 cells transiently expressing the transporter to be examined, to assess the effects of myricetin (100 µM) on the uptake of folate by the PCFTs and riboflavin by hRFVT3. For hPCFT, myricetin was confirmed to induce a transient inhibition and also a sustained inhibition. However, myricetin induced neither transient nor sustained type of rPCFT inhibition. hRFVT3 was inhibited by myricetin in a transient manner, but not in a sustained manner. These results suggest the involvement of a hPCFT-specific mechanism in the sustained inhibition. The transient inhibition may be induced by a mechanism specific to hPCFT and also hRFVT3.

Functional Identification of Plasma Membrane Monoamine Transporter (PMAT/SLC29A4) as an Atenolol Transporter Sensitive to Flavonoids Contained in Apple Juice.

The intestinal absorption of atenolol has recently been reported to be reduced by simultaneous ingestion of fruit juices, such as apple juice. This finding implies a possibility that an unidentified carrier-mediated transport system, which could be interfered by some components of those juices, might be involved in atenolol absorption. In an attempt to explore that possibility, we successfully identified plasma membrane monoamine transporter (PMAT/SLC29A4) as a transporter that can operate for cellular atenolol uptake in the intestine, using Madin-Darby canine kidney II cells stably expressing PMAT. The specific uptake of atenolol by PMAT was greatest at around pH 6.0 and decreased with an increase in pH. At pH 6.0, the PMAT-specific uptake of atenolol was saturable with a Michaelis constant of 0.907 mM. Moreover, PMAT-specific atenolol uptake was extensively inhibited by phloretin and quercetin, which are the major flavonoids contained in apple juice, with the half maximal inhibitory concentrations of 33.3 and 116.3 µM, respectively. PMAT-specific atenolol uptake was also inhibited by several ß-blockers, suggesting that they may also be recognized and transported by PMAT. These results suggest that PMAT is an atenolol transporter that may be involved in intestinal atenolol absorption and sensitive to flavonoids contained in apple juice.

Role of Equilibrative Nucleobase Transporter 1/SLC43A3 as a Ganciclovir Transporter in the Induction of Cytotoxic Effect of Ganciclovir in a Suicide Gene Therapy with Herpes Simplex Virus Thymidine Kinase.

A suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) with ganciclovir (GCV) has been under development as a tumor-targeted therapy; however, the mechanism of cellular GCV uptake, which is prerequisite in the therapy, has not been clarified. In an attempt to resolve this situation and gain information to optimize HSV-TK/GCV system for cancer therapy, we found that human equilibrative nucleobase transporter 1 (ENBT1) can transport GCV with a Michaelis constant of 2.75 mM in Madin-Darby canine kidney II (MDCKII) cells stably transfected with this transporter. In subsequent experiments using green fluorescent protein (GFP)-tagged ENBT1 (GFP-ENBT1) and HSV-TK, the uptake of GCV (30 µM), which was minimal in MDCKII cells and unchanged by their transfection with HSV-TK alone, was increased extensively by their transfection with GFP-ENBT1, together with HSV-TK. Accordingly, cytotoxicity, which was assessed by the WST-8 cell viability assay after the treatment of those cells with GCV (30 µM) for 72 hours, was induced in those transfected with GFP-ENBT1, together with HSV-TK but not in those transfected with HSV-TK alone. These results suggest that ENBT1 could facilitate GCV uptake and thereby enhance cytotoxicity in HSV-TK/GCV system. We also identified Helacyton gartleri (HeLa) and HepG2 as cancer cell lines that are rich with ENBT1 and A549, HCT-15 and MCF-7 as those poor with ENBT1. Accordingly, the HSV-TK/GCV system was effective in inducing cytotoxicity in the former but not in the latter. Thus, ENBT1 was found to be a GCV transporter that could enhance the performance of HSV-TK/GCV suicide gene therapy.

Characteristic Analysis of Intestinal Transport in Enterocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

We previously demonstrated that differentiated enterocytes from human induced pluripotent stem (iPS) cells exhibited drug-metabolizing activities and cytochrome P450 CYP3A4 inducibility. The aim of this study was to apply human iPS cell-derived enterocytes in pharmacokinetic studies by investigating the characteristics of drug transport into enterocyte-like cells. Human iPS cells cultured on feeder cells were differentiated into endodermal cells using activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, epidermal growth factor and small-molecule compounds induced the maturation of the intestinal stem cell-like cells. After differentiation, we performed transepithelial electrical resistance (TEER) measurements, immunofluorescence staining, and transport studies. TEER values increased in a time-dependent manner and reached approximately 100 Ω × cm(2) Efflux transport of Hoechst 33342, a substrate of breast cancer resistance protein (BCRP), was observed and inhibited by the BCRP inhibitor Ko143. The uptake of peptide transporter 1 substrate glycylsarcosine was also confirmed and suppressed when the temperature was lowered to 4°C. Using immunofluorescence staining, villin and Na(+)-K(+) ATPase were expressed. These results suggest that human iPS cell-derived enterocytes had loose tight junctions, polarity, as well as uptake and efflux transport functions. In addition, the rank order of apparent membrane permeability coefficient (Papp) values of these test compounds across the enterocyte-like cell membrane corresponded to the fraction absorbance (Fa) values. Therefore, differentiated enterocytes from human iPS cells may provide a useful comprehensive evaluation model of drug transport and metabolism in the small intestine.

Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals.

The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na(+) and H(+), but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes.

Kinetic and time-dependent features of sustained inhibitory effect of myricetin on folate transport by proton-coupled folate transporter.

Myricetin is a flavonoid that has recently been suggested to induce sustained inhibition of proton-coupled folate transporter (PCFT/SLC46A1), which operates for intestinal folate uptake. The present study was conducted to characterize the inhibitory effect in more detail, using human PCFT stably expressed in Madin-Darby canine kidney II cells, to gain information to cope with problems potentially arising from that. The kinetics of saturable folate transport was first assessed in the absence of myricetin in the cells pretreated with the flavonoid for 60 min. The pretreatment induced PCFT inhibition in a manner dependent on the concentration of myricetin, where the maximum transport rate was reduced by 35.5% and 83.1%, respectively, at its concentrations of 20 µM and 50 µM. The inhibitory effect was, however, less extensive at lower folate concentrations, because the Michaelis constant was also reduced similarly in a manner dependent on myricetin concentration. The inhibition was induced depending on the time of pretreatment and, after removal of myricetin (50 µM) upon the manifestation of an extensive inhibition at 60 min, reversed almost completely in 90 min. This rather short time required for recovery may suggest that the sustained inhibition of PCFT is of a reversible type.

Sustained inhibition of proton-coupled folate transporter by myricetin.

Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled folate transporter (PCFT), the uptake of folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 µM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of folate uptake, which was attributable to a reduction in the maximum transport rate (Vmax). Although the affinity of the transporter for folate was increased at the same time as indicated by a reduction in the Michaelis constant (Km), the change in Km was overwhelmed in extent by that in Vmax. Consistent with the finding, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in Vmax and Km by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of folate and also antifolate drugs.

Functional identification of organic cation transporter 1 as an atenolol transporter sensitive to flavonoids.

Atenolol, a ß1-adrenergic receptor blocker, is administered orally and its intestinal absorption has recently been indicated to be mediated by carrier protein and reduced markedly by ingestion of some fruit juices, such as apple and orange juices. This could be postulated to be a problem arising from the interaction of some components of fruit juices with atenolol at a transporter involved in its intestinal uptake, but the responsible transporter and its interacting components have not been identified yet. In an attempt to examine that possibility, we could successfully find that human organic cation transporter 1 (OCT1/SLC22A1), which is suggested to be expressed at the brush border membrane of enterocytes, is highly capable of transporting atenolol. In this attempt, OCT1 was stably expressed in Madin-Darby canine kidney II cells and the specific uptake of atenolol by the transporter was found to be saturable, conforming to the Michaelis-Menten kinetics with the maximum transport rate (Vmax) of 4.00 nmol/min/mg protein and the Michaelis constant (Km) of 3.08 mM. Furthermore, the OCT1-specific uptake was found to be inhibited by various flavonoids, including those contained in fruit juices that have been suggested to interfere with intestinal atenolol absorption. Particularly, phloretin and quercetin, which are major components of apple juice, were potent in inhibiting OCT1-mediated atenolol transport with the inhibition constants of 38.0 and 48.0 µM, respectively. It is also notable that the inhibition by these flavonoids was of the noncompetitive type. These results indicate that OCT1 is an atenolol transporter that may be involved in intestinal atenolol uptake and sensitive to fruit juices, although its physiological and clinical relevance remains to be further examined.

Noncompetitive inhibition of proton-coupled folate transporter by myricetin.

Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. The present study was conducted to examine that possibility, focusing on its inhibitory effect on proton-coupled folate transporter (PCFT) as the molecular entity of the transport system. The uptake transport of folate was first examined in the Caco-2 cell as an intestinal epithelial cell model, and its carrier-mediated component, of which the Michaelis constant (Km) was 0.407 µM, was found to be noncompetitively inhibited by myricetin with an inhibition constant (Ki) of 61 µM. Consistent with that, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II (MDCKII) cells, of which the Km was 1.246 µM, was also noncompetitively inhibited by myricetin with a Ki of 130 µM. Thus, myricetin was suggested to inhibit intestinal folate transport by acting noncompetitively on PCFT, although the Km and Ki were similarly shifted to some extent to be smaller in Caco-2 cells. Finally, epigallocatechin-3-gallate was also suggested to act in a noncompetitive manner as an inhibitory flavonoid. Care may need to be taken, therefore, in the ingestion of myricetin and some flavonoids to maintain the absorption of folate and antifolate drugs.

Competitive inhibition of AQP7-mediated glycerol transport by glycerol derivatives.

Aquaporin 7 (AQP7) is an aquaglyceroporin that has recently been found to operate as a facilitative carrier rather than a channel for glycerol, although its primary function is as a water channel. To probe into its substrate specificity, we examined the inhibitory effect of a series of acyl glycerol derivatives on glycerol transport mediated by human AQP7 stably expressed in Madin-Darby canine kidney II cells. According to kinetic analyses, AQP7-mediated glycerol transport was found to be competitively inhibited by monoacetin, monobutyrin and diacetin. Therefore, it may be possible that they all could be recognized as substrates by AQP7. The inhibition constant (Ki) of monoacetin (134 µM) was smaller than that of diacetin (420 µM), but greater than the Michaelis constant for glycerol (11.8 µM). Considering another finding that inhibition by triacetin was insignificant, it is likely that a decrease in the number of hydroxyl groups in the glycerol molecule by acetyl derivatization leads to a decrease in affinity for AQP7. The Ki of monobutyrin (80 µM) was, on the other hand, comparable with that of monoacetin, suggesting that the extension of the acyl chain by two hydrocarbon units does not have an impact on affinity for AQP7.

Functional characteristics of aquaporin 7 as a facilitative glycerol carrier.

Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for glycerol as well as for water. However, our recent studies have indicated that AQP9 and AQP10 operate in a carrier mode, which is of saturable nature, for glycerol transport. Assuming that such a functional characteristic could also be shared by AQP7, another aquaglyceroporin, we examined its glycerol transport function. The specific transport of glycerol by human AQP7, which was stably expressed in Madin-Darby canine kidney II cells, was indeed highly saturable, indicating the involvement of a carrier mode of operation mechanism. Kinetic analysis indicated that the specific transport conformed to Michaelis-Menten kinetics with the Michaelis constant of 11.9 µM and was not associated with a nonsaturable transport component as an indication of a simultaneous channel mode of operation, which was previously indicated for AQP10. AQP7-specific glycerol transport was furthermore found to be specifically inhibited by several compounds analogous to glycerol and operate without requiring either Na(+) or H(+). These characteristics of the carrier mode of AQP7 operation suggest that it is a facilitative carrier for glycerol and, possibly, also for analogous compounds, providing a novel insight into its operation mechanism.

Transcriptional regulation of PCFT by KLF4, HNF4α, CDX2 and C/EBPα: implication in its site-specific expression in the small intestine.

Proton-coupled folate transporter (PCFT), which is responsible for the intestinal uptake of folates and analogs, is expressed only in the proximal region in the small intestine. The present study was to examine its transcriptional regulation, which may be involved in such a unique expression profile and potentially in its alteration, using dual-luciferase reporter assays in human embryonic kidney (HEK) 293 cells. The luciferase activity derived from the reporter construct containing the 5'-flanking sequence of -1695/+96 of the human PCFT gene was enhanced most extensively by the introduction of Krüppel-like factor 4 (KLF4). The KLF4-induced luciferase activity was further enhanced by hepatocyte nuclear factor 4α (HNF4α) synergistically. To the contrary, caudal-type homeobox transcription factor 2 (CDX2) and CCAAT/enhancer-binding protein α (C/EBPα) extensively suppressed the luciferase activity induced by KLF4 alone and also that induced by KLF4 and HNF4α. Western blot analysis using the rat small intestine indicated uniform expression of KLF4 along the intestinal tract, proximal-oriented expression of HNF4α, distal-oriented expression of CDX2 and C/EBPα. These results suggest that the activity of PCFT promoter is basically induced by KLF4 and the gradiented expression profile of PCFT may be at least in part accounted for by those of HNF4α, CDX2 and C/EBPα.