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Congenital tooth agenesis is one of the most common anomalies in humans. Many genetic factors are involved in tooth development, including MSX1, PAX9, WNT10A, and LRP6. Thus, mutations in these genes can cause congenital tooth agenesis in humans. In this study, we identified a novel nonsense WNT10A variant, NM_025216.3(WNT10A_v001):c.1090A > T, which produces a C-terminal truncated gene product, p.(Lys364*), in a sporadic form of congenital tooth agenesis. The variant was not found in the healthy parents and thus was considered to cause congenital tooth agenesis in the case.

Psychological stress plays a major role in depression, and interleukin-6 (IL-6) is elevated during depression and psychological stress. MicroRNAs (miRNAs) in extracellular vesicles (EVs), including exosomes and microvesicles, suppress mRNA expression in other cells when endocytosed. In this study, we analyzed the effect of IL-6 on EVs secreted by neural precursor cells. Cells from the human immortalized neural precursor cell line LUHMES were treated with IL-6. EVs were collected using a nanofiltration method. We then analyzed the uptake of LUHMES-derived EVs by astrocytes (ACs) and microglia (MG). Microarray analysis of miRNAs was performed using EV-incorporated RNA and intracellular RNA from ACs and MG to search for increased numbers of miRNAs. We applied the miRNAs to ACs and MG, and examined the cells for suppressed mRNAs. IL-6 increased several miRNAs in the EVs. Three of these miRNAs were originally low in ACs and MG (hsa-miR-135a-3p, hsa-miR-6790-3p, and hsa-miR-11399). In ACs and MG, hsa-miR-6790-3p and hsa-miR-11399 suppressed four mRNAs involved in nerve regeneration (NREP, KCTD12, LLPH, and CTNND1). IL-6 altered the types of miRNAs in EVs derived from neural precursor cells, by which mRNAs involved in nerve regeneration were decreased in ACs and MG. These findings provide new insights into the involvement of IL-6 in stress and depression.

We conducted intratracheal instillations of polyacrylic acid (PAA) with crosslinking and non-crosslinking into rats in order to examine what kinds of physicochemical characteristics of acrylic-acid-based polymers affect responses in the lung. F344 rats were intratracheally exposed to similar molecular weights of crosslinked PAA (CL-PAA) (degree of crosslinking: ~0.1%) and non-crosslinked PAA (Non-CL-PAA) at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months, and 6 months post-exposure. Both PAAs caused increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 6 months following instillation. The release of lactate dehydrogenase (LDH) activity in the BALF was higher in the CL-PAA-exposed groups. Histopathological findings of the lungs demonstrated that the extensive fibrotic changes caused by CL-PAA were also greater than those in exposure to the Non-CL- PAA during the observation period. CL-PAA has more fibrogenicity of the lung, suggesting that crosslinking may be one of the physicochemical characteristic factors of PAA-induced lung disorder.

Background: We conducted intratracheal instillations of different molecular weights of polyacrylic acid (PAA) into rats in order to examine what kinds of physicochemical characteristics of acrylic acid-based polymer affect responses in the lung. Methods: F344 rats were intratracheally exposed to a high molecular weight (HMW) of 598 thousand g/mol or a low molecular weight (LMW) of 30.9 thousand g/mol PAA at low and high doses. Rats were sacrificed at 3 days, 1 week, 1 month, 3 months and 6 months post exposure. Results: HMW PAA caused persistent increases in neutrophil influx, cytokine-induced neutrophil chemoattractants (CINC) in the bronchoalveolar lavage fluid (BALF), and heme oxygenase-1 (HO-1) in the lung tissue from 3 days to 3 months and 6 months following instillation. On the other hand, LMW PAA caused only transient increases in neutrophil influx, CINC in BALF, and HO-1 in the lung tissue from 3 days to up to 1 week or 1 month following instillation. Histopathological findings of the lungs demonstrated that the extensive inflammation and fibrotic changes caused by the HMW PAA was greater than that in exposure to the LMW PAA during the observation period. Conclusion: HMW PAA induced persistence of lung disorder, suggesting that molecular weight is a physicochemical characteristic of PAA-induced lung disorder.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that β-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by β-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

This work studies the relationship between lung inflammation caused by nanomaterials and surfactant protein D (SP-D) kinetics and investigates whether SP-D can be a biomarker of the pulmonary toxicity of nanomaterials. Nanomaterials of nickel oxide and cerium dioxide were classified as having high toxicity, nanomaterials of two types of titanium dioxides and zinc oxide were classified as having low toxicity, and rat biological samples obtained from 3 days to 6 months after intratracheal instillation of those nanomaterials and micron-particles of crystalline silica were used. There were different tendencies of increase between the high- and low-toxicity materials in the concentration of SP-D in bronchoalveolar-lavage fluid (BALF) and serum and in the expression of the SP-D gene in the lung tissue. An analysis of the receiver operating characteristics for the toxicity of the nanomaterials by SP-D in BALF and serum showed a high accuracy of discrimination from 1 week to 3 or 6 months after exposure. These data suggest that the differences in the expression of SP-D in BALF and serum depended on the level of lung inflammation caused by the nanomaterials and that SP-D can be biomarkers for evaluating the pulmonary toxicity of nanomaterials.

Zinc‐finger protein 143 (ZNF143) is a transcription factor that is involved in anticancer drug resistance and cancer cell survival. In the present study, we identified a novel small molecule N‐(5‐bromo‐2‐methoxyphenyl)‐3‐(pyridine‐3‐yl) propiolamide (YPC‐21661) that inhibited ZNF143 promoter activity and down‐regulated the expression of the ZNF143‐regulated genes, RAD51, PLK1, and Survivin, by inhibiting the binding of ZNF143 to DNA. In addition, YPC‐21661 was cytotoxic and induced apoptosis in the human colon cancer cell line, HCT116 and human prostate cancer cell line, PC‐3. 2‐(pyridine‐3‐ylethynyl)‐5‐(2‐(trifluoromethoxy)phenyl)‐1,3,4‐oxadiazole (YPC‐22026), a metabolically stable derivative of YPC‐21661, induced tumor regression accompanied by the suppression of ZNF143‐regulated genes in a mouse xenograft model. The present study revealed that the inhibition of ZNF143 activity by small molecules induced tumor regression in vitro and in vivo; therefore, ZNF143 is a promising target of cancer therapeutics.

We investigated the effects of bicarbonate/lactate-buffered peritoneal dialysis fluid (B/L-PDF) and lactate-buffered PDF (L-PDF) on cell viability and apoptosis, focusing on monocarboxylate transporters (MCTs). MCT-1 transports lactate into cells. Cell viability and apoptosis of human peritoneal mesothelial cells (HPMCs) were examined by water-soluble tetrazolium salt-1 and TUNEL assays, respectively. The relative number of viable HPMCs was significantly decreased by L-PDF at 48 h (8.8 ± 0.4%) compared with cells cultured in M199, but not by B/L-PDF (66.7 ± 1.1%). Apoptosis was markedly induced by L-PDF at 48 h (69.3 ± 16.2%), but not by B/L-PDF (2.6 ± 0.3%). Knockdown of MCT-1 by small interfering RNA (siRNA) attenuated the L-PDF-induced reduction of viable cells and increased apoptosis compared with control siRNA, but MCT-4 knockdown had no effect. B/L-PDF had lesser effects on cell viability and apoptosis of HPMCs compared with L-PDF. These results suggest that B/L-PDF biocompatibility occurs by avoiding the induction of apoptosis in HPMCs.

The lectin Dectin-1 is a good target for β-glucan-mediated drug delivery. Although many murine studies of Dectin-1 have been performed, its human analog has not been studied well in terms of being a drug delivery target. We thus analyzed human Dectin-1 cDNA obtained from chronic myelogenous leukemia-derived cells, CML-1, and confirmed the findings of previous studies that there are many isoforms of human Dectin-1 due to exon skipping, although murine Dectin-1 has only two forms. When we transfected the Dectin-1 gene into a non-Dectin-1-expressing cell line and examined cellular uptake of the antisense DNA/β-glucan complex, we confirmed that expression of the target gene was effectively suppressed through β-glucan/Dectin-1-mediated uptake. The present results suggest that the β-glucan complex would be an effective tool to deliver antisense oligonucleotide (AS-ODN) to Dectin-1-expressing cells not only for mice but also for humans.

Aurora kinase B (AURKB) inhibitors are regarded as potential molecular-targeting drugs for cancer therapy. The present study evaluated the cytotoxic effect of a combination of AZD1152-hQPA, an AURKB inhibitor, and various anticancer agents on the HeLa human cervical cancer cell line, as well as its cisplatin-resistant equivalent HCP4 cell line. It was demonstrated that AZD1152-hQPA had an antagonistic effect on the cytotoxicity of cisplatin, etoposide and doxorubicin, but had a synergistic effect on that of all-trans-retinoic acid (ATRA), Am80 and TAC-101, when tested on HeLa cells. Cisplatin, etoposide and doxorubicin were shown to increase the cellular expression of AURKB, while ATRA, Am80 and TAC-101 downregulated its expression. These results suggested that AURKB expression is regulated by these anticancer agents at the transcriptional level, and that the level of expression of AURKB may influence the cytotoxic effect of AZD1152-hQPA. Therefore, when using anticancer agents, decreasing the expression of AURKB using a molecular-targeting drug may be an optimal therapeutic strategy.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a β-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the β-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the β-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Background We have reported that WNT10A plays a critical role in the growth of fibroblasts/myofibroblasts and microvascular endothelial cells, i.e.; wound healing/scarring. To ascertain the in vivo regulatory, central functions of WNT10A, we examined the net effects of WNT10A depletion using WNT10A-deficient mice (WNT10A–/–). Methods and results We generated WNT10A–/–mice, displaying a range of unique phenotypes of morpho/organogenetic failure, such as growth retardation, alopecia, kyphosis and infertility, and then focused on the functions of WNT10A in wound healing. We subjected C57BL/6J wild-type (WT) or WNT10A–/–mice to skin ulcer formation. The WNT10A–/–mice had significantly larger injured areas and delayed wound healing, which were associated with (a) a smaller number of fibroblasts/myofibroblasts and microvessels; and (b) more reduced expression and synthesis of collagen, compared with WT mice with intact WNT10A expression, especially in those with activated myofibroblasts. Conclusions These observations indicate that WNT10A signaling can play a pivotal in vivo role in wound healing by regulating the expression and synthesis of collagen, as one of fibrogenic factors, at least in part, and critical in vivo roles of WNT10A-mediated effective wound healing are extremely closely associated with collagen expression.

It has been reported that dozens of WNT10A variants are associated with human isolated tooth agenesis, however, little is known about the precise phenotypes. In 50 Japanese patients with severe congenital tooth agenesis, we identified 11 patients with WNT10A variants. Comparing phenotypes between the tooth agenesis patients carrying the wild-type and variants of WNT10A, we revealed that the development of lateral incisors is relatively susceptive to insufficiency of WNT/β-catenin signaling.

Mitochondria are important cellular organelles that function as control centers of the energy supply for highly proliferative cancer cells and regulate apoptosis after cancer chemotherapy. Cisplatin is one of the most important chemotherapeutic agents and a key drug in therapeutic regimens for a broad range of solid tumors. Cisplatin may directly interact with mitochondria, which can induce apoptosis. The direct interactions between cisplatin and mitochondria may account for our understanding of the clinical activity of cisplatin and development of resistance. However, the basis for the roles of mitochondria under treatment with chemotherapy is poorly understood. In this review, we present novel aspects regarding the unique characteristics of the mitochondrial genome in relation to the use of platinum-based chemotherapy and describe our recent work demonstrating the importance of the mitochondrial transcription factor A (mtTFA) expression in cancer cells.

The SMARCE1 (SWI / SNF-related, matrix-associated, and actin-dependent regulator of chromatin, subfamily e, member 1) encodes BAF57 protein. Previously, we reported that BAF57 is a predictive marker of endometrial carcinoma. In this study, we investigated BAF57 expression in ovarian cancer cell lines and their sensitivities to cisplatin, doxorubicin, paclitaxel, and 5-fluorouracil. BAF57 expression was strongly correlated with sensitivities to cisplatin, doxorubicin, and 5-fluorouracil in 10 ovarian cancer cell lines. Paclitaxel sensitivity was also correlated with BAF57 expression, but without significance. In A2780 ovarian cancer cells, knockdown of BAF57 using specific siRNA increased cell cycle arrest at G1 phase and the sensitivities to these anticancer agents. cDNA microarray analysis of A2780 cells transfected with BAF57 siRNA showed that 134 genes were positively regulated by BAF57, including ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2) encoding breast cancer resistance protein (BCRP). We confirmed that knockdown of BAF57 decreased BCRP expression in ovarian cancer cells by Western blot analysis, and that ABCG2 gene expression might be regulated transcriptionally. These results suggested that BAF57 is involved in ovarian cancer cell growth and sensitivity to anticancer agents, and that BAF57 may be a target for ovarian cancer therapy.

Inhalation studies are the gold standard for the estimation of the harmful effects of respirable chemical substances, while there is limited evidence of the harmful effects of chemical substances by intratracheal instillation. We reviewed the effectiveness of intratracheal instillation studies for estimating the hazards of nanoparticles, mainly using papers in which both inhalation and intratracheal instillation studies were performed using the same nanoparticles. Compared to inhalation studies, there is a tendency in intratracheal instillation studies that pulmonary inflammation lasted longer in the lungs. A difference in pulmonary inflammation between high and low toxicity nanoparticles was observed in the intratracheal instillation studies, as in the inhalation studies. Among the endpoints of pulmonary toxicity, the kinetics of neutrophil counts, percentage of neutrophils, and chemokines for neutrophils and macrophages, heme oxygenase-1 (HO-1) in bronchoalveolar lavage fluid (BALF), reflected pulmonary inflammation, suggesting that these markers may be considered the predictive markers of pulmonary toxicity in both types of study. When comparing pulmonary inflammation between intratracheal instillation and inhalation studies under the same initial lung burden, there is a tendency that the inflammatory response following the intratracheal instillation of nanoparticles is greater than or equal to that following the inhalation of nanoparticles. If the difference in clearance in both studies is not large, the estimations of pulmonary toxicity are close. We suggest that intratracheal instillation studies can be useful for ranking the hazard of nanoparticles through pulmonary inflammation.

NiO nanoparticles were administered to rat lungs via intratracheal instillation or inhalation. During pulmonary toxicity caused by NiO nanoparticles, the induction of oxidative stress is a major factor. Both intratracheal instillation and inhalation of NiO nanoparticles induced pulmonary oxidative stress. The oxidative stress response protein, heme oxygenase-1 (HO-1), was induced by the administration of NiO nanoparticles at both the protein and gene expression level. Additionally, certain oxidative-stress markers in the lung, such as 8-iso-prostaglandin F2α, thioredoxin, and inducible nitric oxide synthase were increased. Furthermore, the concentration of myeloperoxidase (MPO) in the lung was also increased by the administration of NiO nanoparticles. When the amount of NiO in the lung is similar, the responses against pulmonary oxidative stress of intratracheal instillation and inhalation are also similar. However, the state of pulmonary oxidative stress in the early phase was different between intratracheal instillation and inhalation, even if the amount of NiO in the lung was similar. Inhalation causes milder oxidative stress than that caused by intratracheal instillation. On evaluation of the nanoparticle-induced pulmonary oxidative stress in the early phase, we should understand the different states of oxidative stress induced by intratracheal instillation and inhalation.

Mitochondrial transcription factor A (mtTFA) is one of the high mobility group protein family and is required for both transcription from and maintenance of mitochondrial genomes. However, the roles of mtTFA have not been extensively studied in cancer cells. Here, we firstly reported the nuclear localization of mtTFA. The proportion of nuclear-localized mtTFA varied among different cancer cells. Some mtTFA binds tightly to the nuclear chromatin. DNA microarray and chromatin immunoprecipitation assays showed that mtTFA can regulate the expression of nuclear genes. Overexpression of mtTFA enhanced the growth of cancer cell lines, whereas downregulation of mtTFA inhibited their growth by regulating mtTFA target genes, such as baculoviral IAP repeat-containing 5 (BIRC5; also known as survivin). Knockdown of mtTFA expression induced p21-dependent G1 cell cycle arrest. These results imply that mtTFA functions in both nuclei and mitochondria to promote cell growth.

Peroxiredoxin 4 (PRDX4) is one of a newly discovered family of antioxidative proteins. We generated human PRDX4 (hPRDX4) transgenic (Tg) mice, displaying a high level of hPRDX4 expression in the pancreatic islets, and then focused on the functions of PRDX4 in a type 1 diabetes mellitus (T1DM) model using a single high dose of streptozotocin (SHDS). After SHDS-injection, Tg mice showed significantly less hyperglycemia and hypoinsulinemia and a much faster response on glucose tolerance test than wild-type (WT) mice. Morphologic and immunohistochemical observation revealed that the pancreatic islet areas of Tg mice were larger along with less CD3-positive lymphocyte infiltration compared with WT mice. Upon comparison between these two mouse models, β-cell apoptosis was also repressed, and reversely, β-cell proliferation was enhanced in Tg mice. Real-time RT-PCR demonstrated that the expression of many inflammatory-related molecules and their receptors and transcription factors were significantly downregulated in Tg mice. These data indicate that PRDX4 can protect pancreatic islet β-cells against injury caused by SHDS-induced insulitis, which strongly suggests that oxidative stress plays an essential role in SHDS-induced diabetes. This study, for the first time, implicates that PRDX4 has a pivotal protective function against diabetes progression in this T1DM model.

Passage of transcribed U snRNA precursors through Cajal bodies ensures that they are properly bound to the PHAX adaptor protein required for nuclear exit., Phosphorylated adaptor for RNA export (PHAX) is the key export mediator for spliceosomal U small nuclear RNA (snRNA) precursors in metazoa. PHAX is enriched in Cajal bodies (CBs), nuclear subdomains involved in the biogenesis of small ribonucleoproteins. However, CBs’ role in U snRNA export has not been demonstrated. In this study, we show that U snRNA precursors microinjected into Xenopus laevis oocyte nuclei temporarily concentrate in CBs but gradually decrease as RNA export proceeds. Inhibition of PHAX activity by the coinjection of a specific anti-PHAX antibody or a dominant-negative PHAX mutant inhibits U snRNA export and simultaneously enhances accumulation of U snRNA precursors in CBs, indicating that U snRNAs transit through CBs before export and that binding to PHAX is required for efficient exit of U snRNAs from CBs. Similar results were obtained with U snRNAs transcribed from microinjected genes. These results reveal a novel function for CBs, which ensure that U snRNA precursors are properly bound by PHAX.

Histone acetyltransferase (HAT) regulates transcription. We have previously shown that two HAT genes, Clock and Tip60, are overexpressed, and upregulate glutathione biosynthesis and the expression of DNA repair genes in cisplatin-resistant cells. To better understand the mechanism of HAT-related drug resistance, we investigated the role of another HAT gene, p300/CBP-associated factor (PCAF), and found that PCAF was also overexpressed in cisplatin-resistant cells and endowed an antiapoptotic phenotype through enhanced E2F1 expression. PCAF-overexpressing cells showed enhanced expression of E2F1 and conferred cell resistance to chemotherapeutic agents. Downregulation of PCAF decreased E2F1 expression and sensitized cells to chemotherapeutic agents. Moreover, knockdown of PCAF induced G1 arrest and apoptosis. These results suggest that PCAF is one of pleiotropic factors for drug resistance and seems to be critical for cancer cell growth. Mol Cancer Res; 8(6); 864–72. ©2010 AACR.

We have recently reported that N-myc downstream-regulated gene 1 (NDRG1)/Ca(2+)-associated protein with a molecular mass of 43kDa (Cap43) suppresses angiogenesis and tumor growth of pancreatic cancer through marked decreases in both the expression of CXC chemokines and phosphorylation of a NF-kappaB signaling molecule, inhibitor of kappaB kinase (IkappaBalpha). NDRG1/Cap43 is phosphorylated at serine/threonine sites in its C-terminal domain by serum- and glucocorticoid-regulated kinase 1 (SGK1). In this study, we attempted to clarify the domain or site of NDRG1/Cap43 responsible for its suppression of CXC chemokine expression in pancreatic cancer cells. Expression of the deletion constructs CapDelta2 [deletion of amino acids (AA) 130-142] and CapDelta4 [deletion of AA 180-294] as well as the wild-type full sequence of NDRG1/Cap43 (F-Cap), suppressed the production of CXC chemokines such as Groalpha/CXCL1 and ENA-78/CXCL5, whereas no or low suppression was observed in cell expressing the CapDelta5 mutant [deletion of AA 326-350] and CapDelta6 mutant [deletion of AA 326-394]. We further introduced mutations at the serine and threonine sites at 328 [T328A], 330 [S330A] and 346 [T346A], which are susceptible to phosphorylation by SGK1, and also constructed double mutants [T328A, S330A], [T328A, T346A] and [S330A, T346A]. Expression of all these mutants, with the exception of [S330A, T346A], suppressed the production of CXC chemokine to similar levels as their wild-type counterpart. IkappaBalpha was found to be specifically phosphorylated by this double mutant [S330A, T346A] and the CapDelta5 mutant at levels comparable to that induced in their wild-type counterpart. Phosphorylation of NDRG1/Cap43 at both serine330 and threonine346 is required for its suppressive action on the NF-kappaB signaling pathway and CXC chemokine expression in pancreatic cancer cells.

Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mitochondrial DNA. This study was conducted to elucidate the clinicopathologic and prognostic significance of mtTFA in patients with endometrial carcinoma. This study investigated the relationship between the immunohistochemical expression of mtTFA and various clinicopathological variables in 276 endometrial carcinomas, including 245 endometrioid adenocarcinomas and 31 nonendometrioid carcinomas (21 serous carcinomas and 10 clear cell adenocarcinomas). Both uni- and multivariate regression analyses were performed. The mtTFA labeling index of endometrioid adenocarcinomas ranged from 0% to 98%, with a median value of 32%, which was selected as the cut-off point for mtTFA expression. The mtTFA expression in endometrioid adenocarcinomas was significantly associated with the surgical stage, myometrial invasion, lymphovascular space invasion, cervical invasion, and lymph node metastasis. In contrast, no correlation between clinicopathologic variables and mtTFA expression was found in nonendometrioid carcinomas. Correlation analysis between mtTFA and p53 expression by using the Pearson test showed significant correlation in endometrioid adenocarcinomas (P = 0.007), but no significant correlation in nonendometrioid carcinomas (P = 0.947). A univariate survival analysis showed that the 10-year overall survival rate of the patients with mtTFA-positive endometrioid adenocarcinoma was significantly worse than that of patients with mtTFA-negative endometrioid adenocarcinoma (80.8% vs. 93.8%, P = 0.012). However, the multivariate analysis revealed that mtTFA expression in endometrioid adenocarcinomas was no independent prognostic factor. The positive mtTFA expression is a useful maker for progression of the tumors and the poor prognosis of the patients in endometrioid adenocarcinomas.

The hazards of various types of nanoparticles with high functionality have not been fully assessed. We investigated the usefulness of biopersistence as a hazard indicator of nanoparticles by performing inhalation and intratracheal instillation studies and comparing the biopersistence of two nanoparticles with different toxicities: NiO and TiO2 nanoparticles with high and low toxicity among nanoparticles, respectively. In the 4-week inhalation studies, the average exposure concentrations were 0.32 and 1.65 mg/m3 for NiO, and 0.50 and 1.84 mg/m3 for TiO2. In the instillation studies, 0.2 and 1.0 mg of NiO nanoparticles and 0.2, 0.36, and 1.0 mg of TiO2 were dispersed in 0.4 mL water and instilled to rats. After the exposure, the lung burden in each of five rats was determined by Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES) from 3 days to 3 months for inhalation studies and to 6 months for instillation studies. In both the inhalation and instillation studies, NiO nanoparticles persisted for longer in the lung compared with TiO2 nanoparticles, and the calculated biological half times (BHTs) of the NiO nanoparticles was longer than that of the TiO2 nanoparticles. Biopersistence also correlated with histopathological changes, inflammatory response, and other biomarkers in bronchoalveolar lavage fluid (BALF) after the exposure to nanoparticles. These results suggested that the biopersistence is a good indicator of the hazards of nanoparticles.

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

YB-1 controls gene expression through both transcriptional and translational mechanisms and is involved in various biological activities such as brain development, chemoresistance, and tumor progression. We have previously shown that YB-1 is overexpressed in cisplatin-resistant cells and is involved in resistance against DNA-damaging agents. Structural analysis of the YB-1 promoter reveals that several E-boxes may participate in the regulation of YB-1 expression. Here, we show that the E-box–binding transcription factor Twist is overexpressed in cisplatin-resistant cells and that YB-1 is a target gene of Twist. Silencing of either Twist or YB-1 expression induces G1 phase cell cycle arrest of tumor cell growth. Significantly, reexpression of YB-1 led to increase colony formation when Twist expression was down-regulated by small interfering RNA. However, cotransfection of Twist expression plasmid could not increase colony formation when YB-1 expression was down-regulated. Collectively, these data suggest that YB-1 is a major downstream target of Twist. Both YB-1 and Twist expression could induce tumor progression, promoting cell growth and driving oncogenesis in various cancers. Thus, both YB-1 and Twist may represent promising molecular targets for cancer therapy. [Cancer Res 2008;68(1):98–105]

Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.

The eukaryotic Y-box-binding protein-1 (YB-1) is involved in the transcriptional and translational control of many biological processes, including cell proliferation. In clinical studies, the cellular level of YB-1 closely correlates with tumor growth and prognosis. To understand the role of YB-1 in vivo, especially in the developmental process, we generated YB-1 knock-out mice, which are embryonic lethal and exhibit exencephaly associated with abnormal patterns of cell proliferation within the neuroepithelium. beta-Actin expression and F-actin formation were reduced in the YB-1 null embryo and YB-1(-/-) mouse embryonic fibroblasts, suggesting that the neural tube defect is caused by abnormal cell morphology and actin assembly within the neuroepithelium. Fibroblasts derived from YB-1(-/-) embryos demonstrated reduced growth and cell density. A colony formation assay showed that YB-1(-/-) mouse embryonic fibroblasts failed to undergo morphological transformation and remained contact-inhibited in culture. These results demonstrate that YB-1 is involved in early mouse development, including neural tube closure and cell proliferation.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

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.

Cisplatin is one of the most potent and widely used anti-cancer agents in the treatment of various solid tumors. However, the development of resistance to cisplatin is a major obstacle in clinical treatment. Several mechanisms are thought to be involved in cisplatin resistance, including decreased intracellular drug accumulation, increased levels of cellular thiols, increased nucleotide excision-repair activity and decreased mismatch-repair activity. In general, the molecules responsible for each mechanism are upregulated in cisplatin-resistant cells; this indicates that the transcription factors activated in response to cisplatin might play crucial roles in drug resistance. It is known that the tumor-suppressor proteins p53 and p73, and the oncoprotein c-Myc, which function as transcription factors, influence cellular sensitivity to cisplatin. So far, we have identified several transcription factors involved in cisplatin resistance, including Y-box binding protein-1 (YB-1), CCAAT-binding transcription factor 2 (CTF2), activating transcription factor 4 (ATF4), zinc-finger factor 143 (ZNF143) and mitochondrial transcription factor A (mtTFA). Two of these-YB-1 and ZNF143-lack the high-mobility group (HMG) domain and can bind preferentially to cisplatin-modified DNA in addition to HMG domain proteins or DNA repair proteins, indicating that these transcription factors may also participate in DNA repair. In this review, we summarize the mechanisms of cisplatin resistance and focus on transcription factors involved in the genomic response to cisplatin.

The expression of the PDGF β-receptor is tightly regulated during a normal cell cycle. c-Myc and p73α repress transcription of the receptor through interaction with NF-Y. In ST15A cells which stably express the temperature-sensitive SV40 large T antigen (LT) the receptor expression and ligand binding decreased under the permissive condition. Transient expression of the LT, but not small t, decreased the endogenous receptor expression at mRNA and protein levels in NIH3T3 cells but not in the myc-null HO15.19 cells. The wild-type LT, but not the various pRb or p53 binding defective LT mutants, represses the PDGF β-receptor promoter activity. Moreover, the inability of the LT-mediated repression in the myc-null cells, the Rb-null 3T3 cells, and the Saos-2 cells lacking pRb and p53, indicates that Myc, pRb and p53 are all necessary elements. PDGF β-receptor promoter-luciferase assays revealed that the CCAAT motif is important for the repression. Furthermore, p53 was found to increase the promoter activity mainly via the upstream Sp1 binding sites together with the CCAAT motif in the NIH 3T3 cells. This was confirmed by Schneider's Drosophila line (SL2) cells deficient in both endogenous NF-Y and Sp1. Chromatin immunoprecipitation using ST15A cells revealed that both LT and p53 bound the PDGF β-receptor promoter and the binding of p53 diminished when LT was expressed in the permissive condition. However, LT binds the promoter in the absence of pRb and p53 in Saos-2 cells stably expressing LT. These results suggest that LT binds the promoter and interferes with NF-Y and Sp1 to repress it in the presence of Myc, pRb and p53.

The C-terminus of p53 is responsible for maintaining the latent, non-DNA-binding form of p53. However, the mechanism by which the C-terminus regulates DNA binding is not yet fully understood. We show here that p53 interacts with RNA via its C-terminal domain and that disruption of this interaction, by RNase A treatment, truncation or phosphorylation of the C-terminus, restores DNA-binding activity. Furthermore, the oligomerization of p53 is significantly enhanced by disrupting the interaction between p53 and RNA. These findings suggest that binding of RNA to p53 is involved in the mechanism of p53 latency.

We have identified a cisplatin-inducible gene, the mitochondrial ribosomal protein S11 (MRP S11) gene, by means of mRNA differential display. Functional analysis of the MRP S11 promoter showed that a Staf binding site in the promoter is required for both basal promoter activity and cisplatin-inducible activity. We also found that Staf binding activity is significantly increased in nuclear extracts from cells treated with cisplatin. ZNF 143 and ZNF 76 are human homologues of the Xenopus transcriptional activator, Staf. ZNF 143 expression is induced by cisplatin but ZNF 76 expression is not. However, ZNF 143 expression is not induced by transplatin, which is clinically ineffective. ZNF143 is an inducible gene by other DNA damaging agents such as γ-irradiation, etoposide and adriamycin. ZNF 143 also binds preferentially to cisplatin-modified DNA. These results suggest that ZNF 143 participates in cellular responses to DNA damage. © 2004 Wiley-Liss, Inc.

We demonstrated recently that expression of the UDP-N-acetyl-α-d-galactosamine: polypeptide N-acetylgalactosaminyltrans-ferase-3 (GalNAc-T3) gene is restricted to epithelial glands [Nomoto, Izumi, Ise, Kato, Takano, Nagatani, Shibao, Ohta, Imamura, Kuwano, Matsuo, Yamada, Itoh and Kohno (1999) Cancer Res. 59, 6214–6222]. In the present study, we show that sodium butyrate treatment of human breast cancer MCF-7 cells transcriptionally activates the GalNAc-T3 gene. Transient transfection of plasmids containing a reporter gene under the control of GalNAc-T3 indicated that several transcriptional elements are involved in response to sodium butyrate, with the nuclear respiratory factor-1 (NRF-1)-binding motif located between −88 and −77nt being the most important. Incubation of a labelled probe encompassing the NRF-1-binding motif with a nuclear extract of sodium butyrate-treated MCF-7 cells yielded a higher level of specific DNA–protein complex versus controls. Flag-tagged NRF-1 expressed in MCF-7 cells can bind to the NRF-1-binding motif of the GalNAc-T3 promoter. Nuclear content of NRF-1 remained constant in MCF-7 cells treated with or without sodium butyrate. Moreover, NRF-1 interacts with and is acetylated by p300/CBP-associated factor (P/CAF). Acetylation of NRF-1 enhances DNA binding. Co-transfection of the GalNAc-T3 reporter plasmid with either NRF-1 or P/CAF expression plasmid resulted in the activation of the GalNAc-T3 promoter. These results indicate a correlation between acetylation of NRF-1 by P/CAF and the butyrate-induced expression of the GalNAc-T3 gene. Additionally, induced expression of P/CAF may be a component of the adenocarcinoma differentiation process.

The vacuolar-ATPase (V-ATPase) is a multi-subunit enzyme that couples ATP hydrolysis to proton pumping across membranes. V-ATPase genes are considered to be housekeeping genes and are expressed in human neoplastic tissue and in cell lines. We have isolated and characterized several genomic clones containing the 5′-end of the human V-ATPase genes. DNA sequence analysis of the promoters of two V-ATPase subunit genes, encoding C ( ATP6C ) and cʺ ( ATP6F ), reveals GC-rich regions in the region of the first exon. Neither TATA- nor CCAAT-boxes were found in these promoters, but both GC-boxes and E-boxes were identified. Transient transfection analysis, using a series of 5′ nested deletions of promoter–luciferase constructs in human cancer cells, demonstrated that a positive cis-acting regulatory region was present in these TATA-less promoters. The regions between −79 and −40 of the ATP6C promoter and between −245 and −99 of the ATP6F promoter were identified as being likely to be extremely important for basal promoter activity. Electrophoretic mobility shift assays (EMSA) of these cis-regulatory regions revealed the basal promoter to be highly complex, with cooperative binding of several transcription factors, including Sp family members. These data identify the critical regulatory regions for both the ATP6C and ATP6F basal promoters and stress the functional importance of multiple protein complexes, involving the Sp family of transcription factors, in regulating gene expression.

The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication. Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication. The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes. In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73α up-regulates the activity of this promoter. Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73α-induced p21 promoter activity. Using deletion mutants, we found that the DNA-binding domains of both p53 and p73α are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively. CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73α. These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.

We recently reported that c-Myc represses the transcription of platelet-derived growth factor (PDGF) beta-receptor (Izumi, H., Molander, C., Penn, L. Z., Ishisaki, A., Kohno, K., and Funa, K. (2001) J. Cell Sci. 114, 1533-1544). We demonstrate here that the p53 family protein p73alpha represses PDGF beta-receptor transcription essentially by the same mechanism. p73alpha but not p73beta or p53 represses the transcription in concordance with its ability to bind NF-YC and NF-YB. None of other p73 isoforms (i.e. p73beta, p73gamma, p73epsilon), C-terminal deletion mutants of p73alpha, and p53 is able to bind NF-Y with the exception of p63alpha. This finding suggests that the sterile alpha-motif domain present only in p73alpha and p63alpha is the interaction site. For the repression, the N-terminal transactivation domain of p73alpha is also indispensable, arguing for the importance of the activity of p73alpha in the mechanism. p73alpha binds the C-terminal HAP domain of NF-YC previously found to be the interaction site with c-Myc and TBP. Because c-Myc induces and activates p73alpha (Zaika, A., Irwin, M., Sansome, C., and Moll, U. M. (2001) J. Biol. Chem. 276, 11310-11316) and they bind each other (Uramoto, H., Izumi, H., Ise, T., Tada, M., Uchiumi, T., Kuwano, M., Yasumoto, K., Funa, K., and Kohno, K. (2002) J. Biol. Chem. 277, in press), we examined whether the repression by p73 is dependent on c-Myc. However, Myc-null rat fibroblasts are also susceptible to p73alpha-induced repression. Serum stimulation of NIH3T3 cells gradually decreased the amount of endogenous NF-Y binding to the PDGF beta-receptor promoter, whereas NF-YA expression in the nuclear extracts remains unchanged. Our results indicate that serum stimulation induces c-Myc and p73alpha, leading to the down-regulation of PDGF beta-receptor expression by repressing its transcription.

YB-1 is a member of the cold shock domain family of proteins that is important for signaling DNA damage and cell proliferation. YB-1 is induced by DNA damage and can also recognize cisplatin-modified DNA. In this study we observed a 6-fold increase in the steady-state level of YB-1 mRNA in response to cisplatin exposure in cells of the human cancer cell line KB. We present evidence from cotransfection experiments for a critical role of c-Myc and p73 in the transactivation of the YB-1 promoter. p73 transactivated the YB-1 promoter in experiments with Saos-2 cells, which express c-Myc, but not with HO15.19 cells, which lack c-Myc. In turn, c-Myc transactivated an intact YB-1 promoter but not a YB-1 promoter with a mutant E-box, indicating that the E-box is necessary for the response of the promoter to cisplatin. We also found that p73 interacts with c-Myc in vitro and in vivo. Using deletion mutants we showed that the DNA-binding domain of p73 and the C-terminal region of c-Myc are required for the interaction. Furthermore, p73 stimulated the interaction of Max with c-Myc and promoted binding of the c-Myc-Max complex to its target DNA. Our data suggest that p73 stimulates the transcription of the YB-1 promoter by enhancing recruitment of the c-Myc-Max complex to the E-box.

Objectives We have previously reported that decreased expression of PTEN in lung cancer PC9 cells harboring an EGFR-activating mutation (del E746–A750) results in acquisition of resistance to EGFR-TKIs, gefitinib and erlotinib, accompanied by enhanced phosphorylation of Akt and decreased nuclear translocation of a transcription factor EGR-1 [8] . In the present study, PTEN promoter methylation accounted for the decreased expression of PTEN in our gefitinib-resistant mutant. Material and methods DNA methylation status of the PTEN promoter in PC9 and gefitinib-resistant cells were examined using methylation-specific PCR. The effect of DNA methylation on PTEN expression was evaluated by treatment of lung cancer cell lines with 5-aza-2′-deoxycytidine (5AZA-CdR). Results We observed the characteristics of two gefitinib-resistant sublines, GEF1-1 and GEF2-1, derived from PC9 as follows. (1) PTEN overexpression suppressed AKT phosphorylation and restored the sensitivity to gefitinib and erlotinib in GEF1-1 cells. (2) EGR-1 siRNA mediated knockdown suppressed the expression of cyclin D1 and ICAM-1 genes but not of PTEN gene in PC9 cells. (3) Transfection of EGR-1 cDNA into a drug-resistant subline induced the expression of cyclin D1 and ICAM-1 but not of PTEN. (4) Treatment with 5AZA-CdR induced the expression of PTEN in resistant sublines but not in the parental line PC9. (5) A CpG site near the translational start point of the 5′-regulatory region was methylated in GEF1-1 and GEF2-1 but not in PC9. Conclusion Our results strongly suggest that CpG hypermethylation of the PTEN gene contributes to the decreased expression of PTEN during acquired resistance to gefitinib or erlotinib.

c-Myc plays a key role in the cell cycle dependent control of the PDGF (β)-receptor mRNA. The mouse platelet-derived growth factor (PDGF) (β)-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF (β)-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF (β)-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.

The mouse PDGF β-receptor promoter is tightly controlled by NF-Y that binds to a CCAAT box located upstream of the initiation site [1, 2]. In this report, we show that Sp1 plays an essential role in the PDGF β-receptor transcription. Within the upstream GC rich area there are two Sp1 binding sites located in close proximity to the CCAAT box. Deletion of the GC rich region resulted in a 50% decrease of the transcriptional activity of the promoter, and a complete loss of its responsiveness to over-expression of Sp1. There was an additive effect between NF-Y and Sp1 in reporter activity when they were co-transfected together with the promoter-reporter construct. Furthermore, transfection of NF-Y failed to enhance transcriptional activity when the Sp1 binding sites were deleted from the promoter, suggesting an important role for Sp1 in this NF-Y controlled transcription. We have recently reported that c-Myc represses PDGF β-receptor transcription through its interference with the transactivation activity of NF-Y [3]. In the case of p21waf1/cip1 transcription, c-Myc was shown to repress its transcription by sequestering Sp1 [4]. However, we could not find any effect of Sp1 in the c-Myc-mediated repression on the PDGF β-receptor promoter, since the deletion of Sp1 binding sites could not attenuate the repression by c-Myc on the promoter activity.

The Y-box binding protein, YB-1, belongs to a family of multifunctional proteins which regulate gene expression on both transcriptional and translational levels. The tumor suppressor gene p53 displays growth suppressive properties by regulating gene expression through transcriptional regulation. We now demonstrate that YB-1 directly interacts with p53 using an in vitro pull-down assay. Using immunochemical co-precipitation methods, we also found that the two proteins are bound in vivo. Deletion analysis showed that three independent domains of YB-1, one at the N-terminal and two at the C-terminal, interact with p53. Conversely, a 14 amino acid sequence at the C-terminal of p53 was required for its interaction with YB-1. Gel mobility shift assays showed that the interaction of YB-1 with p53 stimulated the sequence-specific DNA binding of p53 to its consensus sequence. By contrast, this interaction inhibited the binding of YB-1. Using a p53-responsive p21 promoter linked to a reporter gene, it can be shown that antisense expression of YB-1 inhibits the induction of this promoter by p53 in transient transfection assays. These findings delineate a straightforward mechanism for gene expression through p53-YB-1 interaction.

PDGF and TNF-alpha are both known to play important roles in inflammation, albeit frequently by opposing actions. Typically, TNF-alpha can attenuate PDGF beta-receptor signaling. Pretreatment of mouse 3T3 L1 fibroblasts with TNF-alpha greatly diminished their proliferative response to PDGF. However, TNF-alpha affected neither the binding of PDGF-BB to cell surface receptors nor the total amount of PDGF beta-receptor in the cells, but decreased the PDGF-induced in vitro kinase activity of the receptor. The phosphatase inhibitor ortho-vanadate did not prevent this effect. Ortho-phosphate labeling of cells prior to TNF-alpha treatment and PDGF-BB stimulation confirmed a decrease of in vivo phosphorylation of the PDGF beta-receptor. Two-dimensional mapping after tryptic cleavage as well as phosphoamino acid analysis demonstrated a general decrease in phosphorylation of all known tyrosine residues in the PDGF beta-receptor. The exact mechanism for this suppression remains to be clarified (Less)

The transcription factor YB-1 is expressed in a wide range of cell types and has been implicated in the regulation of various genes involved in cell proliferation. Nuclear expression of YB-1 is correlated with MDR-1 gene expression in breast cancer and osteosarcoma. In this study, we asked whether YB-1 expression is enhanced in human colorectral carcinoma and if it is associated with the expression of target genes such as MDR-1, DNA topoisomerase II alpha and PCNA. YB-1, DNA topoisomerase II alpha, PCNA and MDR-1 expression were assessed by Western blotting, Northern blotting and immunohistochemistry in 26 human colorectal carcinomas. The involvement of YB-1 in DNA topoisomerase II alpha gene expression was examined by transient DNA transfection assays. YB-1 was overexpressed in almost all cancerous lesions in comparison with normal mucosa in surgically resected colorectal carcinomas of 26 patients. YB-1 expression correlated well with both DNA topoisomerase II alpha and PCNA expression. In contrast, no correlation was observed between YB-1 and MDR-1 expression. We also found that a transient co-transfection with a DNA topoisomerase II alpha promoter-luciferase plasmid and an antisense YB-1 expression construct resulted in a significant reduction of the promoter activity in KM12C human colon cancer cells. YB-1 may be an excellent proliferation-associated marker and may be a transcription factor regulating DNA topoisomerase II alpha gene expression in human colorectal carcinoma.

Human cancer cells have been found to express a large number of IL-13 receptors. We have previously shown that mRNA encoding one of these receptors, IL-13Ralpha1, is increased in cisplatin-resistant cells and is upregulated in tumor cells cultured with cisplatin. To understand the molecular mechanism of IL-13Ralpha1 gene expression, we cloned approximately 52 kbp of the IL-13Ralpha1 gene and sequenced the first exon and about 1 kbp of the upstream DNA. The first exon is 211 bp and contains 88 bp of coding sequence, while the first intron is about 13 kbp in length. The promoter region, which is GC rich, was found to lack both TATA and CCAAT boxes. Transient expression assays revealed that transcription of the IL-13Ralpha1 gene was significantly higher in cisplatin-resistant cells than in parental, cisplatin-sensitive cells. Deletion analysis of the IL-13Ralpha1 promoter identified a 70-bp core promoter region upstream of the transcription initiation site. Electrophoretic gel mobility shift assays showed that a synthetic IL-13Ralpha1 oligonucleotide (nt -40 to nt -15) bound a nuclear factor from cisplatin-resistant cells to a significantly greater degree than the equivalent factor from parental cells. This oligonucleotide was found to contain a palindromic sequence with a BstEII recognition site at its center. This palindromic sequence functions to mediate upregulation of IL-13Ralpha1 promoter in cisplatin-resistant cells and deletion or disruption of this sequence also resulted in severe reduction of the promoter activity. These findings suggest that IL-13Ralpha1 expression is upregulated at the transcriptional level in cisplatin-resistant cells. The characterization of both the IL-13Ralpha1 promoter and the transcription factors binding to it may contribute to our understanding of IL-13Ralpha1 regulation in cancer cells.

Transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs) signal via distinct type I and type II receptors and Smad proteins. A nine amino acid sequence between kinase subdomains IV and V in type I receptors, termed the L45 loop, has been shown to be important in conferring signalling specificity. We examined the responses of a mutant TGF-beta type I receptor (TbetaR-I) and a mutant BMPR-IB, in which the L45 regions of these two receptors were exchanged. Swapping the four amino acid residues that are different in BMPR-IB for those in TbetaR-I, and vice versa, switched their type I receptor-restricted Smad activation and specificity in transcriptional responses. These studies identify the L45 loop regions in type I receptors as critical determinants in specifying Smad isoform activation.

The expression of N-myc downstream-regulated gene 1 (NDRG1) was significantly correlated with tumor angiogenesis and malignant progression together with poor prognosis in gastric cancer. However, the underlying mechanism for the role of NDRG1 in the malignant progression of gastric cancer remains unknown. Here we examined whether and how NDRG1 could modulate tumor angiogenesis by human gastric cancer cells. We established NU/Cap12 and NU/Cap32 cells overexpressing NDRG1 in NUGC-3 cells, which show lower tumor angiogenesis in vivo. Compared with parental NU/Mock3, NU/Cap12, and NU/Cap32 cells: 1) induced higher tumor angiogenesis than NU/Mock3 cells accompanied by infiltration of tumor-associated macrophages in mouse dorsal air sac assay and Matrigel plug assay; 2) showed much higher expression of CXC chemokines, MMP-1, and the potent angiogenic factor VEGF-A; 3) increased the expression of the representative inflammatory cytokine, IL-1α; 4) augmented JNK phosphorylation and nuclear expression of activator protein 1 (AP-1). Further analysis demonstrated that knockdown of AP-1 (Jun and/or Fos) resulted in down-regulation of the expression of VEGF-A, CXC chemokines, and MMP-1, and also suppressed expression of IL-1α in NDRG1-overexpressing cell lines. Treatment with IL-1 receptor antagonist (IL-1ra) resulted in down-regulation of JNK and c-Jun phosphorylation, and the expression of VEGF-A, CXC chemokines, and MMP-1 in NU/Cap12 and NU/Cap32 cells. Finally, administration of IL-1ra suppressed both tumor angiogenesis and infiltration of macrophages by NU/Cap12 in vivo. Together, activation of JNK/AP-1 thus seems to promote tumor angiogenesis in relationship to NDRG1-induced inflammatory stimuli by gastric cancer cells.

Patients with end-stage renal disease are exposed to increased oxidative stress and impairment of antioxidant mechanisms. We focused on dialysis renal cell carcinoma (RCC), including epithelial hyperplasia in acquired cystic disease of the kidney (ACDK). We attempted to obtain insight into the carcinogenesis and tumor progression in terms of cellular defense mechanisms associated with oxidative stress by investigating the expression of antioxidant proteins by immunohistochemistry. We evaluated retrospectively 43 cases of dialysis RCC and, as a control group, 49 cases of sporadic RCC. Peroxiredoxin (Prx) 1, 3, 4, 5, and 6 expression in dialysis RCC was positively correlated with the duration of dialysis. In epithelial hyperplasia, in 17 cases of acquired cystic disease of the kidney, Prxs and thioredoxin were highly expressed. Moreover, in dialysis RCC, Prx 3, 4, and 5 immunoreactivity and nuclear expression of Y-box-binding protein-1 were higher than in sporadic RCC. In dialysis RCC, Prx 3, 4, and 5 immunoreactivity positively correlated with the Fuhrman nuclear grade. These data suggest that oxidative stress during dialysis enhances antioxidant activity, with an inhibiting effect on carcinogenesis. Once cancer has developed, antioxidant activity might have a stimulating effect on the progression of dialysis RCC.