Biomedical subjects

Hitoshi Nakagama

Publications and source records attributed to Hitoshi Nakagama.

At least 19 recordsLinked to original sources

PubMed2007

Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells.

Okadaic acid-sensitve serine/threonine protein phosphatase 5 (PP5) is expressed ubiquitously in various tissues and is considered to participate in many cellular processes. PP5 has a catalytic domain in the C-terminal region and three tetratricopeptide repeat (TPR) motifs in the N-terminal region, which are suspected to function as a protein-protein interaction domain. Physiological roles of PP5 are still largely unknown, although several PP5-binding proteins were reported and a few in vivo functions of PP5 were suggested. In the present study, the effects of expression of the full-length wild-type PP5 fused with EGFP (EGFP-PP5(WT)) and its phosphatase-dead mutant EGFP-PP5(H304A) were investigated. Transient expression of either EGFP-PP5(WT) or EGFP-PP5(H304A) in HeLa cells induced deformed nuclei with a 10-fold frequency compared to that of EGFP. Abnormal-shaped nuclei were also substantially increased by induced moderate expression of PP5 in tet-on HeLa cells. Many HeLa cells expressing EGFP-PP5(WT) possessed multi-nuclei separated from each other by nuclear membrane, while expression of EGFP-PP5(H304A) induced deformed nuclei which were multiple-like in shape, but not separated completely and were surrounded by one nuclear membrane. These results suggest that PP5 plays important roles at the M-phase of the cell cycle, especially in separation of chromosomes and formation of nuclear membrane.

Amino Acid Sequence↗

PubMed2007

Effect of running training on DMH-induced aberrant crypt foci in rat colon.

PURPOSE: We examined the effects of treadmill-running training on the induction of aberrant crypt foci (ACF), which is the first step of colon cancer induction, in the colonic mucosa of rats injected with 1,2-dimethylhydrazine (DMH). METHODS: Four-week-old F344 rats (N = 38) were randomly assigned to training (19 rats) and control (19 rats) groups. After a week, all rats were given DMH (20 mg.kg(-1) body weight) once a week for 2 wk. Running training was started at age 7 wk (speed: 10 m.min(-1), 0% grade, 120 min.d(-1), 5 d.wk(-1)). After 4 wk of training, the rats were sacrificed and the colon was removed, opened, and counted for ACF with 0.2% methylene blue staining. RESULTS: Running training resulted in lower body- (P < 0.01) and adipose fat weight (P < 0.05). The numbers of ACF and total AC were significantly lower in the running training group than in the control group (P < 0.05). The occurrences of one, three, and five aberrant crypts per focus were also significantly lower in the running training group than in the control group (P < 0.05). The ratios of total AC/ACF did not significantly differ between the running training and control groups. CONCLUSIONS: The results of the present investigation suggest that low-intensity running training inhibits the DMH-induced initiation of colon ACF development.

Animals↗

PubMed2006

Inhibition of peroxisome proliferator-activated receptor gamma activity in esophageal carcinoma cells results in a drastic decrease of invasive properties.

Esophageal cancer is difficult to treat because of its rapid progression, and more effective therapeutic approaches are needed. The PPARgamma is a nuclear receptor superfamily member that is expressed in many cancers. PPARgamma expression is a feature of esophageal cancer cell lines, and in the present investigation, the PPARgamma antagonists T0070907 and GW9662 could induce loss of invasion but could not induce growth reduction or apoptosis at low concentrations (< 10 mM). A high concentration of antagonists (50 microM) inhibited cell growth and induced apoptosis, but these effects did not explain our result at the low concentration. Morphological change, decreased expression of the cell signaling pathway and inhibition of cancer cell invasion were observed in the low concentration. This suggested that PPARgamma antagonists inhibited esophageal cancer cell invasion as well as cell adherence, most likely due to alteration in the FAK-MAPK pathway, and this was independent of apoptosis. These results suggested that PPARgamma plays an important role in cancer cell invasion and that it might be a novel target for therapy of esophageal cancer.

Actins↗

PubMed2006

Predominant T helper type 2-inflammatory responses promote murine colon cancers.

Colon cancer is one of the most serious complications of inflammatory bowel diseases, especially ulcerative colitis (UC). Previous studies have shown that characteristic immunological event during inflammation in UC is the expression of T helper-type 2 (Th2) cell-derived cytokines. In this study, we investigated the influence of a predominant Th2-type cytokine response in colitis on carcinogen-induced colon tumors. Wild type (WT), interferon gamma (IFN-gamma) gene deficient (-/-) [Th2 dominant] or interleukin (IL)-4(-/-) [Th1-dominant] mice of BALB/c background were used in this study. To compare tumor formation, mice were given the carcinogen azoxymethane (AOM) and intrarectal administration of trinitrobenzene sulfonic acid (TNBS), to induce colitis. Thirty-three weeks after initial treatment, the total colon was examined. When IFN-gamma(-/-) mice were treated with AOM and TNBS, significantly higher number of tumors were seen (8.4 +/- 1.7) than in WT (3.3 +/- 2.9) or IL-4(-/-) (3.1 +/- 3.4) mice, which received identical treatments. A separate set of experiment, using less doses of AOM and TNBS also showed the higher frequency of tumor formation in IFN-gamma(-/-) mice than in IL-4(-/-) mice. Histologically, the tumors were well- or moderately-differentiated adenocarcinomas. No invasion into the submucosal or serosal layers of the intestine was seen. In immunohistological staining, some tumors in IFN-gamma(-/-) mice showed distinct nuclear expression of beta-catenin, in contrast to the strong membrane staining seen in tumors of IL-4(-/-) mice. In conclusion, colonic inflammation associated with Th2-dominant cytokine responses enhanced the formation of malignant neoplasms.

Adenocarcinoma↗

PubMed2006

Molecular mechanisms for maintenance of G-rich short tandem repeats capable of adopting G4 DNA structures.

Mammalian genomes contain several types of repetitive sequences. Some of these sequences are implicated in various specific cellular events, including meiotic recombination, chromosomal breaks and transcriptional regulation, and also in several human disorders. In this review, we document the formation of DNA secondary structures by the G-rich repetitive sequences that have been found in several minisatellites, telomeres and in various triplet repeats, and report their effects on in vitro DNA synthesis. d(GGCAG) repeats in the mouse minisatellite Pc-1 were demonstrated to form an intra-molecular folded-back quadruplex structure (also called a G4' structure) by NMR and CD spectrum analyses. d(TTAGGG) telomere repeats and d(CGG) triplet repeats were also shown to form G4' and other unspecified higher order structures, respectively. In vitro DNA synthesis was substantially arrested within the repeats, and this could be responsible for the preferential mutability of the G-rich repetitive sequences. Electrophoretic mobility shift assays using NIH3T3 cell extracts revealed heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and A3, which were tightly and specifically bound to d(GGCAG) and d(TTAGGG) repeats with K(d) values in the order of nM. HnRNP A1 unfolded the G4' structure formed in the d(GGCAG)(n) and d(TTAGGG)(n) repeat regions, and also resolved the higher order structure formed by d(CGG) triplet repeats. Furthermore, DNA synthesis arrest at the secondary structures of d(GGCAG) repeats, telomeres and d(CGG) triplet repeats was efficiently repressed by the addition of hnRNP A1. High expression of hnRNPs may contribute to the maintenance of G-rich repetitive sequences, including telomere repeats, and may also participate in ensuring the stability of the genome in cells with enhanced proliferation. Transcriptional regulation of genes, such as c-myc and insulin, by G4 sequences found in the promoter regions could be an intriguing field of research and help further elucidate the biological functions of the hnRNP family of proteins in human diseases.

3T3 Cells↗

PubMed2006

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding.

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.

14-3-3 Proteins↗

PubMed2006

[Decreased PPARs activity mediated induction of anoikis and inability of cell adhesion and invasion in colorectal carcinoma cells].

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Animals↗

PubMed2005

Parp-1 deficiency does not increase the frequency of tumors in the oral cavity and esophagus of ICR/129Sv mice by 4-nitroquinoline 1-oxide, a carcinogen producing bulky adducts.

The impact of poly(ADP-ribose) polymerase-1 (Parp-1)-deficiency on 4-nitroquinoline 1-oxide (4NQO)-induced carcinogenesis was studied in mice with an ICR/129Sv mixed genetic background. Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) animals given 4NQO for thirty-two weeks at 0.001% in their drinking water developed papillomas and squamous cell carcinomas of the tongue, palate and esophagus, but with no statistically significant variation with the Parp-1 genotype. Thus Parp-1 deficiency does not elevate susceptibility to carcinogenesis induced by a carcinogen which gives rise to bulky DNA lesions. This study also indicated that the ICR/129Sv mixed genetic background is associated with high yield induction of esophageal tumors by 4NQO.

4-Nitroquinoline-1-oxide↗

PubMed2005

Strain differences in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon carcinogenesis in mice.

We have recently developed a mouse model for colitis-related colon carcinogenesis by a combined treatment with azoxymethane (AOM) and dextran sodium sulfate (DSS) in male ICR mice. However, strain differences in the sensitivity to AOM/DSS-induced colon carcinogenesis in mice have yet to be elucidated. The aim of this study was to determine the presence of any genetically determined differences in sensitivity to our model of colon carcinogenesis in four inbred strains of mice. Male Balb/c, C3H/HeN, C57BL/6N and DBA/2N mice were given a single intraperitoneal injection of AOM (10 mg/kg body wt), followed by 1% DSS (w/v) in drinking water for 4 days, and thereafter they received no further treatment for up to 16 weeks. At the end of the study (Week 18), all mice were killed and a histopathological analysis of their colon was performed. The incidence of colonic adenocarcinoma was 100% with a multiplicity (no. of tumors/mouse) of 7.7+/-4.3 in the Balb/c mice and 50% with a multiplicity of 1.0+/-1.2 in the C57BL/6N mice. On the other hand, only a few colonic adenomas, but no adenocarcinomas, developed in the C3H/HeN mice (29% incidence with a multiplicity of 0.7+/-1.5) and the DBA/2N mice (20% incidence with a multiplicity of 0.2+/-0.4). The inflammation and immunohistochemical nitrotyrosine-positivity scores of the mice treated with AOM and DSS in the decreasing order were as follows: C3H/HeN>Balb/c>DBA/2N>C57BL/6N and Balb/c>C57BL/6N>C3H/HeN>DBA/2N, respectively. Our results thus indicated the presence of strain differences in the susceptibility to AOM/DSS-induced colonic tumorigenesis. These differences may have been directly influenced by the response to nitrosation stress due to the inflammation caused by DSS.

Adenocarcinoma↗

PubMed2005

Parp-1 deficiency does not enhance liver carcinogenesis induced by 2-amino-3-methylimidazo[4,5-f]quinoline in mice.

The susceptibility of poly(ADP-ribose) polymerase-1 (Parp-1) knockout mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced liver carcinogenesis was analyzed. Twelve-week-old male Parp-1(+/+), Parp-1(+/-) and Parp-1(-/-) mice of the C57BL/6 congenic strain were fed a diet containing IQ at a concentration of 300 ppm or a control diet for 60 weeks. Hepatocellular carcinomas were observed only in 1/19, 2/18 and 1/17 of the Parp-1(-/-), Parp-1(+/-) and Parp-1(+/+) mice, respectively. Parp-1 deficiency did not affect the susceptibility of mice to carcinogenicity of IQ, which produces bulky DNA adducts that are repaired mainly through the nucleotide excision repair pathway. This result is in sharp contrast to the increased susceptibility of Parp-1(-/-) mice to carcinogenesis induced by alkylating agents that produce DNA damage repaired mainly through base excision repair and DNA strand break repair pathways.

Adenoma, Liver Cell↗

PubMed2005

Differential staining of dysplastic aberrant crypt foci in the colon facilitates prediction of carcinogenic potentials of chemicals in rats.

We developed a novel and simple method to identify dysplastic aberrant crypt foci (ACF) induced in rats by colon carcinogens more efficiently and selectively without conducting laborious histological examination, which usually requires enough time to get final diagnosis. By adding a simple decolorization process with 70% methanol after conventional 0.2% methylene blue staining, dysplastic ACF could be differentially contrasted. To examine the validity of this novel method, which we refer to as differential staining, we analyzed colonic lesions induced by three heterocyclic amines, including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and found that the number of dysplastic ACF detected more precisely reflected their carcinogenic potential than the total numbers of ACF.

Animals↗

PubMed2005

Parp-1 deficiency causes an increase of deletion mutations and insertions/rearrangements in vivo after treatment with an alkylating agent.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P<0.05). Categorization of the mutations revealed that deletions larger than 1 kb or those accompanying 1-5 bp insertions at the deletion junctions, as well as rearrangements, were more frequently observed in Parp-1-/- than in Parp-1+/+ mice (P<0.05, respectively). In contrast, mutant frequencies of the gpt gene in the livers of Parp-1(-/-) and Parp-1(+/+) mice after BHP treatment were both elevated and there was no significant difference between the genotypes. These results indicate that Parp-1 is implicated in suppressing deletion mutations in vivo, especially those accompanying small insertions or rearrangements.

Alkylating Agents↗

PubMed2005

Genetic alteration of poly(ADP-ribose) polymerase-1 in human germ cell tumors.

Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5'-UTR (untranslated region) were also detected.

Amino Acid Sequence↗

PubMed2005

Modeling human colon cancer in rodents using a food-borne carcinogen, PhIP.

Animal models provide researchers with powerful tools to elucidate multistage mechanisms for cancer development and to gain further insights into the biological roles of various cancer-related genes in in vivo situations. As for colon cancer models in rodents, Apc-disrupted mice, including ApcMin, have been one of the most widely utilized animal models to dissect the molecular events implicated in the development of intestinal tumors. In rats, several models have been established using chemical carcinogens, including azoxymethane and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP). The former is a representative colon carcinogenic alkylating agent, and the latter a heterocyclic amine produced while cooking meat and fish, which people are exposed to in ordinary life. It is of great importance to note that PhIP preferentially targets the colon and prostate gland in male rats, and the mammary glands in female rats. Cancers in these three organs are common in Western countries and are currently increasing in Japan, where modern dietary habits are rapidly becoming more like those of the West. In the present article, the history of PhIP-induced colon cancer models in rodents, activation/detoxification mechanisms of PhIP with regard to the formation of PhIP-DNA adducts, mechanistic approaches to dissect the molecular events involved in the development of colon cancer by PhIP, and epidemiological evidence of human exposure to PhIP are overviewed. The induction of Paneth cell maturation/differentiation in PhIP-induced colon cancers, genetic traits affecting susceptibility to colon carcinogenesis, and the biological relevance of colon cancer models in rodents to studying human colon carcinogenesis are also discussed.

Animals↗

PubMed2005

Unfolding of higher DNA structures formed by the d(CGG) triplet repeat by UP1 protein.

Fragile X syndrome is caused by expansion of a d(CGG) triplet repeat in the 5'-untranslated region of the first exon of the FMR1 gene resulting in silencing of the gene. The d(CGG) repeat has been reported to form hairpin and quadruplex structures in vitro, and formation of these higher structures could be responsible for its unstable expansion in the syndrome, although molecular mechanisms underlying the repeat expansion still remain elusive. We have previously proved that UP1, a proteolytic product of hnRNP A1, unfolds the intramolecular quadruplex structures of d(GGCAG)5 and d(TTAGGG)4 and abrogates the arrest of DNA synthesis at d(GGG)n sites. Here, we demonstrate that the d(CGG) repeat forms a peculiar DNA structure, which deviates from the canonical B-form structure. In addition, UP1 was demonstrated by CD spectrum analysis to unfold this characteristic higher structure of the d(CGG) repeat and to abrogate the arrest of DNA synthesis at the site. This ability of UP1 suggests that unfolding of unusual DNA structures of a triplet repeat is required for DNA synthesis processes.

Circular Dichroism↗

PubMed2005

Up-regulation of hnRNP A1 gene in sporadic human colorectal cancers.

We have previously reported that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a major hnRNP, binds to G-rich repetitive sequences and quadruplex (G4') structures in DNA, including the 5'-TTAGGG-3' telomere repeat and 5'-GGCAG-3' short-tandem-repeat. DNA synthesis arrest at the (GGG) sites within these repeats in vitro was retrieved by the addition of the hnRNP A1 protein or its N-terminal proteolytic product, UP1, in a dose-dependent manner. Therefore, functional perturbation of hnRNP A1 may abrogate the genomic stability of telomere repeats and other G-rich sequences, independent of its major role in transcriptional and translational regulation. In the present study, we conducted genetic and expression analysis of the hnRNP A1 gene in sporadic human colorectal cancers to clarify its possible involvement in human carcinogenesis. Of 30 lesions, one harbored a mutation at the -11 position from the translation initiation site, but none in the coding region. A single nucleotide polymorphism, an A or G-allele, was found in the 5' upstream promoter region of the gene. Quantitative gene expression analysis revealed that 60% (18/30) of cases showed over-expression of hnRNP A1 in cancer tissues by 2-fold or greater, compared to their normal colon tissues, with values of 78, 64 and 40% for clinicopathological stages II, III and IV, respectively. Although the biological consequences of hnRNP A1 overexpression in colorectal cancers remain to be clarified, it could contribute to maintenance of telomere repeats in cancer cells with enhanced cell proliferation. Alternatively, since the variations in the stoichiometry of hnRNP family proteins are considered to affect cell-specific gene expression, quantitative alteration of hnRNP A1 could result in facilitation of transformation of colon epithelial cells as a consequence of transcriptional and translational perturbation.

Adult↗

PubMed2004

Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.

The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.

14-3-3 Proteins↗

PubMed2004

LRP130, a single-stranded DNA/RNA-binding protein, localizes at the outer nuclear and endoplasmic reticulum membrane, and interacts with mRNA in vivo.

LRP130 (also known as a LRPPRC) is an RNA and single-stranded DNA-binding protein, and recently identified as a candidate gene responsible for the Leigh syndrome, a French-Canadian type cytochrome c oxidase deficiency. However, the biological function of LRP130 still remains largely unresolved. In the present study, we found that the C-terminal half of the mouse LRP130 located within a 120 amino acid sequence (a.a. 845-964) binds to synthetic RNA homopolymers, poly(G), poly(U), and poly(C), as well as r(CUGCC)(6). Assessment of the subcellular localization indicated both nuclear/endoplasmic reticulum (ER) and mitochondrial fractions to be positive. To further analyze the subcellular localization of LRP130, a nuclear/ER fraction was fractionated into the nucleoplasm (NP) and nuclear envelope (NE)/ER, and the latter was further separated into outer nuclear membrane (ONM)/ER and inner nuclear membrane (INM) by treatment with Triton X-100. LRP130 was detectable in all three fractions, and the distribution pattern was in good accordance with that known for ONM/ER proteins. Interestingly, immunostaining of HeLa cells demonstrated nuclear rim staining of LRP130, specifically at the outside of the NE and also at ER, and association of LRP130 with poly(A)(+) RNA was restricted only to the ONM/ER fraction. Overexpression of full-length mouse LRP130 fused with EGFP resulted in nuclear accumulation of poly(A)(+) RNA in HeLa cells. Taking all these results together, it is suggested that LRP130, a novel type of RNA-binding protein, associates with mRNA/mRNP complexes at the outside of NE and ER, and plays a role in control of mRNA metabolisms.

Animals↗