MUTYH, an adenine DNA glycosylase, mediates p53 tumor suppression via PARP-dependent cell death.

p53-regulated caspase-independent cell death has been implicated in suppression of tumorigenesis, however, the regulating mechanisms are poorly understood. We previously reported that 8-oxoguanine (8-oxoG) accumulation in nuclear DNA (nDNA) and mitochondrial DNA triggers two distinct caspase-independent cell death through buildup of single-strand DNA breaks by MutY homolog (MUTYH), an adenine DNA glycosylase. One pathway depends on poly-ADP-ribose polymerase (PARP) and the other depends on calpains. Deficiency of MUTYH causes MUTYH-associated familial adenomatous polyposis. MUTYH thereby suppresses tumorigenesis not only by avoiding mutagenesis, but also by inducing cell death. Here, we identified the functional p53-binding site in the human MUTYH gene and demonstrated that MUTYH is transcriptionally regulated by p53, especially in the p53/DNA mismatch repair enzyme, MLH1-proficient colorectal cancer-derived HCT116+Chr3 cells. MUTYH-small interfering RNA, an inhibitor for p53 or PARP suppressed cell death without an additive effect, thus revealing that MUTYH is a potential mediator of p53 tumor suppression, which is known to be upregulated by MLH1. Moreover, we found that the p53-proficient, mismatch repair protein, MLH1-proficient colorectal cancer cell line express substantial levels of MUTYH in nuclei but not in mitochondria, suggesting that 8-oxoG accumulation in nDNA triggers MLH1/PARP-dependent cell death. These results provide new insights on the molecular mechanism of tumorigenesis and potential new strategies for cancer therapies.

ITPase-deficient mice show growth retardation and die before weaning.

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

Galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the dentate gyrus of adult mouse hippocampus.

We examined the expression of galectin-1, an endogenous lectin with one carbohydrate-binding domain, in the adult mouse hippocampus after systemic kainate administration. We found that the expression of galectin-1 was remarkably increased in activated astrocytes of the CA3 subregion and dentate gyrus of the hippocampus, and in nestin-positive neural progenitors in the dentate gyrus. Quantitative reverse transcription PCR (RT-PCR) analysis revealed that the galectin-1 mRNA level in hippocampus began to increase 1 day after kainate administration and that a 13-fold increase was attained within 3 days. Western blotting analysis confirmed that the level of galectin-1 protein increased to more than three-fold a week after the exposure. We showed that isolated astrocytes express and secrete galectin-1. To clarify the significance of the increased expression of galectin-1 in hippocampus, we compared the levels of hippocampal cell proliferation in galectin-1 knockout and wild-type mice after saline or kainate administration. The number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells detected in the subgranular zone (SGZ) of galectin-1 knockout mice decreased to 62% with saline, and to 52% with kainate, as compared with the number seen in the wild-type mice. Most of the BrdU-positive cells in SGZ expressed doublecortin and neuron-specific nuclear protein, indicating that they are immature neurons. We therefore concluded that galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the hippocampus.

Galectin-1beta, a natural monomeric form of galectin-1 lacking its six amino-terminal residues promotes axonal regeneration but not cell death.

We previously identified a novel N-terminally processed form of galectin-1, galectin-1beta (Gal-1beta) whose expression was induced by DeltaFosB. In the present study, the biochemical properties and biological functions of Gal-1beta were compared with the full-length form of galectin-1 (Gal-1alpha). We first purified recombinant mouse Gal-1alpha and beta (rmGal-1alpha, beta) to near homogeneity. The rmGal-1alpha exists as a monomer under oxidized conditions and forms a dimer under reduced conditions, while the rmGal-1beta exists as a monomer regardless of redox conditions. The affinity of rmGal-1beta to beta-lactose was approximately two-fold lower than that of rmGal-1alpha under reduced conditions. The viability of Jurkat cells efficiently decreased when they were exposed to rmGal-1alpha, however, rmGal-1beta barely induced such a reduction. In contrast, both rmGal-1alpha and rmGal-1beta exhibited an equivalent capacity to promote axonal regeneration from the dorsal root ganglion explants. Our results suggest that the biochemical properties of rmGal-1beta determine its biological functions.

DeltaFosB, but not FosB, induces delayed apoptosis independent of cell proliferation in the Rat1a embryo cell line.

The fates of Rat1a cells expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand binding domain of human estrogen receptor were examined. The binding of estrogen to the fusion proteins resulted in their nuclear translocation and triggered cell proliferation, and thereafter delayed cell death was observed only in cells expressing ER-DeltaFosB. The proliferation of Rat1a cells, but not cell death triggered by ER-DeltaFosB, was completely abolished by butyrolactone I, an inhibitor of cycline-dependent kinases, and was partly suppressed by antisense oligonucleotides against galectin-1, whose expression is induced after estrogen administration. The cell death was accompanied by the activation of caspase-3 and -9, the fragmentation of the nuclear genome and cytochrome c release from the mitochondria, and was suppressed by zDEVD-fmk and zLEHD-fmk but not zIETD-fmk. The cell death was not suppressed by exogenous His-PTD-Bcl-x(L) at all, suggesting involvement of a Bcl-x(L)-resistant pathway for cytochrome c release.

Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen.

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.

Role of bile salt hydrophobicity in distribution of phospholipid species to carriers in supersaturated model bile solutions.

BACKGROUND: Phospholipid species modulate cholesterol-holding capacity and, therefore, regulate bile metastability. METHODS: In this study, we investigated the effect of bile salt hydrophobicity on the distribution of phospholipids among lipid particles in supersaturated model bile solutions (total lipid concentration, 9 g/dL; taurocholate/phospholipid ratio 3.0, cholesterol saturation index 1.3), by using gel permeation chromatography. RESULTS: With an increase of bile salt hydrophobicity in the elution buffer, the uptake of cholesterol and phospholipids into bile salt micelles was increased, associated with an increased cholesterol/phospholipid molar ratio of the vesicles. In contrast, there was an inverse correlation between the hydrophobicity of the phospholipid species in the vesicles and that of bile salts in the elution buffer, suggesting that hydrophobic bile salts induced preferential uptake of hydrophobic phospholipids into bile salt micelles, while less hydrophobic phospholipids, with a relatively low cholesterol-holding capacity, remained in the vesicles. CONCLUSIONS: These data indicate that bile salt hydrophobicity regulates vesicular cholesterol metastability by modulating the hydrophobicity of phospholipids in vesicles, as well as the lipid distribution among various biliary lipid particles.

Molecular analysis of suppression of interleukin-8 production by rebamipide in Helicobacter pylori-stimulated gastric cancer cell lines.

Interleukin-8 (IL-8) may play an important role in Helicobacter pylori infection-associated chronic active gastritis and peptic ulcer disease in human. We have recently reported that a gastric cancer cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori. Moreover, H. pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription. In this study, we evaluated the effect of rebamipide, an antigastritis and antiulcer agent, on H. pylori-induced IL-8 production. Rebamipide inhibited the production of IL-8 in several gastric cancer cell lines infected with H. pylori. In addition, rebamipide suppressed H. pylori-induced IL-8 gene expression at the transcriptional level as revealed by northern blotting analysis and luciferase activity in cells that were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44). Furthermore, rebamipide significantly suppressed the NF-kappaB activation by H. pylori infection. These results suggest that rebamipide may protect against the mucosal inflammation associated with H. pylori infection through inhibition of a proinflammatory cytokine, IL-8.

Mechanisms involved in Helicobacter pylori-induced interleukin-8 production by a gastric cancer cell line, MKN45.

Accumulating evidence suggests an important role of interleukin-8 (IL-8) in Helicobacter pylori infection-associated chronic atrophic gastritis and peptic ulcer. We observed in this study that a gastric cancer-derived cell line, MKN45, produced a massive amount of IL-8 upon coculture with live H. pylori but not with killed H. pylori, H. pylori culture supernatants, or live H. pylori separated by a permeable membrane, indicating that IL-8 production requires a direct contact between the cells and live bacteria. Moreover, the tyrosine kinase inhibitor herbimycin but neither a protein kinase C inhibitor (staurosporine) nor a protein kinase A inhibitor (H89) inhibited IL-8 production by MKN45 cells cocultured with live bacteria, suggesting the involvement of a tyrosine kinase(s) in H. pylori-induced IL-8 production. In addition, coculture of H. pylori induced IL-8 mRNA expression in MKN45 cells and an increase in luciferase activity in cells which were transfected with a luciferase expression vector linked with a 5'-flanking region of the IL-8 gene (bp -133 to +44), indicating that the induction of IL-8 production occurred at the transcriptional level. This region contain three cis elements important for induction of IL-8 gene expression: AP-1 (-126 to -120 bp), NF-IL6 (-94 to -81 bp), and NF-kappaB (-80 to -70 bp) binding sites. Mutation of the NF-kappaB binding site abrogated completely the induction of luciferase activity, whereas that of the AP-1 site partially reduced the induction. However, mutation of the NF-IL6 binding site resulted in no decrease in the induction of luciferase activity. Moreover, specific NF-kappaB complexes were detected in the nuclear proteins extracted from MKN45 cells which were infected with H. pylori. Collectively, these results suggest that H. pylori induced the activation of NF-kappaB as well as AP-1, leading to IL-8 gene transcription.

A quantitative assessment of serum chylomicron by light scattering intensity: application to the intestinal fat absorption test.

A novel fat absorption test to clarify the malabsorption syndrome was developed using a micronephelometric technique and compared with the classic conventional technique using 131I-triolein. An integrity of time-sequential light scattered from chylomicron-related turbidity in serum was determined between 0 and 300 min after butter fat load, being expressed in terms of the light scattering intensity (LSI). A good correlation was obtained between LSI and the serum level of chylomicron-triglyceride determined by an ultracentrifugation technique (r = 0.819, P < 0.001). The maximal LSI was consistently observed at 180 min after administration of a test meal in the normal group (n = 39), whereas the malabsorption syndrome group (n = 35) was distinctly different and could be further classified according to four patterns of LSI changes. In addition, an inverse correlation was found between this fat absorption test and the 131I-triolein absorption test. It was concluded that the micronephelometric technique which does not use a radionuclide is advantageous in its simple and safe evaluation of fat malabsorption syndrome.

Effects of myeloid cell growth factors on alkaline phosphatase, myeloperoxidase, defensin and granulocyte colony-stimulating factor receptor mRNA expression in haemopoietic cells of normal individuals and myeloid disorders.

The mRNA expression of alkaline phosphatase (ALP), myeloperoxidase (MPO), defensin and G-CSF receptor (G-CSFR) in bone marrow cells of normal individuals and myeloid disorders, with or without in vitro stimulation by myeloid cell growth factors, i.e. G-CSF, GM-CSF and IL-3, were examined as markers for myeloid cell differentiation in both mononuclear cell (MNC) and polymorphonuclear cell (PMN) fractions. Without any stimulation, ALP mRNA was expressed only in PMNs, G-CSFR mRNA in PMNs were expressed stronger than in MNCs; both MPO and defensin mRNA were expressed to the same degree in both fractions. With stimulation, the ALP mRNA expression in both fractions was strongly enhanced by G-CSF, but the expression was inhibited by GM-CSF and/or IL-3. MPO mRNA expression was stimulated by G-CSF and/or GM-CSF in MNCs. G-CSFR mRNA expression was enhanced by G-CSF in both fractions. Defensin mRNA expression was inhibited by G-CSF. In cases of myelodysplastic syndrome and chronic myelogenous leukaemia which display a suppressed maturation of myeloid cells, our results demonstrated an almost normal response to these growth factors. Our results suggest that studies on these myeloid marker mRNA expressions would provide more knowledge about the differentiation state and cytokine reactivity of myeloid cells in normal individuals as well as various disorders.

Possible correlation between fusion pattern of BCR/ABL mRNA and clinical response to alpha-interferon in chronic myelogenous leukemia.

Twenty six patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) treated with IFN-alpha were classified on the basis of the fusion pattern of BCR/ABL chimeric mRNA determined by a reverse-transcriptase-polymerase chain reaction (RT-PCR) method. The relationship between the fusion pattern of BCR/ABL mRNA and the clinical outcome was also analysed. Twelve patients showed M-bcr exon 3/ABL exon 2 (B3/A2) chimeric mRNA and nine had M-bcr exon 2/ABL exon 2 (B2/A2) mRNA. Eleven of the 12 patients with B3/A2 achieved complete hematological response with IFN-alpha therapy, as did three of the nine patients with B2/A2. The mean duration to blastic crisis was significantly longer in the B3/A2 patients (mean 52.4 months) than in the B2/A2 patients (mean 26.2 months) (p less than 0.01). These results suggest that the fusion pattern of BCR/ABL mRNA may affect the therapeutic response to IFN-alpha and clinical outcome in CML patients.