Imprinting analysis by droplet digital PCR coupled with locked nucleic acid TaqMan probes.

Imprinted genes are differentially expressed in a parent-of-origin-specific manner. Parental origin of the alleles is discriminated by intragenic DNA polymorphisms. Comparisons of parental allelic expression have been analysed by semiquantitative RT-PCR. Here, we developed a novel quantitative method for allelic expression of the imprinted gene Ube3a, which inactivation and mutations cause Angelman syndrome and predominantly expressed by the maternal allele in neuronal tissues. In this method, cDNA was amplified by droplet digital PCR (ddPCR) coupled with allele-specific locked nucleic acid (LNA) TaqMan probes, which labelled by FAM and HEX were designed to detect the SNPs in the target regions. ddPCR assay demonstrated that the sense transcript of Ube3a was equally expressed from both parental alleles in adult tissues except neuronal tissues, where Ube3a expression from the paternal allele was about 10 to 14% of total Ube3a expression in adult brain, and 20% in spinal cord. The antisense transcript of Ube3a was expressed at 60% to 70% of the sense transcript of Ube3a in adult brain. Changes in the Ube3a transcripts during postnatal brain development were also evaluated by ddPCR. The ddPCR method is far more reliable and simpler to use than semiquantitative PCR to analyse skewed or faint allelic expression of imprinted genes.

Transgenic mice with a tandem duplication of the Necdin gene overexpress Necdin.

Necdin (Ndn) transgenic (Tg) mice were generated with a bacterial artificial chromosome (BAC) clone. Droplet digital PCR (ddPCR) and inverse PCR methods revealed that the transgene consisted of four fragments with a total length of 171 kb. Two of these fragments were tandem tail-to-tail duplicates of 77 kb and 37 kb that both contained a Ndn gene. The transgene was inserted in chromosome 15qD1. Ndn is a paternally expressed imprinted gene; however, the total expression level of Ndn in hemizygous Tg mice was approximately twofold higher than that in wild-type mice. ddPCR assays with locked nucleic acid (LNA) TaqMan probes revealed that transgenic Ndn expression was almost equal to endogenous Ndn expression, despite there being two copies of the Ndn gene in the transgene, indicating an interaction between the transcriptional regulation of endogenous Ndn and the transgene. ddPCR assays with LNA TaqMan probes were also applied for imprinting analysis to confirm exclusive paternal expression in tissues with low Ndn expression. This is the first report of a Tg mouse with a tandem duplication of a Ndn transgene and Ndn overexpression, which will be useful for the in vivo study of Ndn overexpression and for rescue experiments of the neonatal lethality seen in the Ndn knockout mouse.

Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts.

Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis. Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model.

Geranylgeraniol oxidase activity involved in oxidative formation of geranylgeranoic acid in human hepatoma cells.

Geranylgeranoic acid (GGA), a 20-carbon acyclic polyprenoic acid (all-trans 3,7,11,15-tetramethyl- 2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were developed as synthetic "acyclic retinoids" for cancer chemoprevention. Previously, we have shown the natural occurrence of GGA in various medicinal herbs and reported enzymatic formation of GGA from geranylgeraniol (GGOH) through geranylgeranial (GGal) by rat liver homogenates. Here, we present several lines of evidence that a putative GGOH oxidase is involved in GGA synthesis by human hepatoma cell lysates. First, conversion of GGOH to GGal did not require exogenous NAD(+), whereas the conversion from GGal to GGA absolutely required additional NAD(+). Second, GGal synthesis from GGOH was coupled with consumption of oxygen from the reaction mixture. Third, GGOH-dependent GGal synthesis activity was proteinase K-resistant and even enhanced by proteinase K treatment; GGOH oxidase activity was enriched in the mitochondrial fraction. Finally, recombinant human monoamine oxidase (MAO)-B, but not MAO-A catalyzed oxidation of GGOH to GGal. These data suggest that a putative mitochondrial GGOH oxidase is involved in the initial step of GGA synthesis from GGOH.

Rapid downregulation of cyclin D1 induced by geranylgeranoic acid in human hepatoma cells.

Geranylgeranoic acid (GGA) and its derivatives are currently under development as chemopreventive agents against second primary hepatoma in Japan. We aimed to evaluate chemoprevention targets of GGA and a surrogate marker of chemopreventive response to clarify the molecular mechanism of hepatoma chemoprevention with GGA. Human hepatoma-derived cell lines such as HuH-7, PLC/PRF/5, and HepG-2, were treated with GGA and its derivatives. Cellular dynamics of several cell-cycle-related proteins were assessed by either immunoblotting or immunofluorescence method. The cellular expression of cyclin D1 protein was suppressed immediately after GGA treatment. This reduction was partially blocked by pretreatment with 26S proteasome inhibitor MG-132, indicating that proteasomal degradation was involved in GGA-induced disappearance of cyclin D1. A phosphorylation of retinoblastoma protein (RB) at serine 780, a target site of cyclin D1-dependent kinase 4, was rapidly decreased in GGA-treated HuH-7 cells. Furthermore, subcellular fractionation, Western blotting, and immunofluorescence revealed GGA-induced nuclear accumulation of RB. These results strongly suggest that cyclin D1 may be a target of chemopreventive GGA in human hepatoma cells. GGA-induced rapid repression of cyclin D1, and a consequent dephosphorylation and nuclear translocation of RB, may influence cell cycle progression and may be relevant to GGA-induced cell death mechanisms.

Polished rice as natural sources of cancer-preventing geranylgeranoic acid.

Geranylgeranoic acid, a 20-carbon polyprenoic acid (all-trans 3,7,11,15-tetramethyl-2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were previously developed as synthetic "acyclic retinoids" for cancer chemoprevention. Recently, we demonstrated the natural occurrence of geranylgeranoic acid in various medicinal herbs (Shidoji and Ogawa, 2004). In this present study, we present several lines of evidence to demonstrate that geranylgeranyl diphosphate taken in foods could be metabolized to GGA through geranylgeraniol and geranylgeranyl aldehyde via the following steps: 1) The conversion from geranylgeranyl diphosphate to geranylgeraniol was demonstrated to occur by the action of bovine intestinal alkaline phosphatase, with a K(m) of 46.1 µM. 2) Geranylgeraniol oxidase-mediated conversion of geranylgeraniol to geranylgeranyl aldehyde was revealed in rat liver homogenates, which activity was mainly localized in the mitochondrial fraction. The mitochondrial enzyme showed a K(m) of 92.9 µM. 3) The conversion of geranylgeranyl aldehyde to geranylgeranoic acid by geranylgeranyl aldehyde dehydrogenase in rat liver homogenates was absolutely dependent on exogenously added NAD(+) or NADP(+). The K(m) of the mitochondrial geranylgeranyl aldehyde dehydrogenase was 27.5 µM for geranylgeranyl aldehyde. Taken together, our data suggest that cancer preventive geranylgeranoic acid could be a physiological metabolite from commonly consumed foods.

Increase in plasma concentrations of geranylgeranoic Acid after turmeric tablet intake by healthy volunteers.

Geranylgeranoic acid (GGA) is one of the most potent cancer-preventive acyclic retinoids. GGA has been shown to induce cell death in human hepatoma-derived HuH-7 cells. We have recently reported the natural occurrence of GGA and its related compounds in several medicinal herbs such as turmeric, basil, rosehip, cinnamon and others [Shidoji and Ogawa, J. Lipid Res., 45: 1092-1103, 2004]. In the present study, we performed oral administration of turmeric tablets to healthy volunteers in order to investigate bioavailability of natural GGA. By using liquid chromatography/mass spectrometry, authentic GGA was eluted at a retention time of around 18 min as a negative ion of m/z 303.4. With healthy volunteers, plasma GGA was detected prior to the tablet intake and its concentrations were increased at 2 h after its intake and maintained at higher level until 4 h, suggesting an efficient bioavailability of preformed GGA in the turmeric tablets through oral administration. These results indicated that GGA in the turmeric tablet was absorbed as an intact form from intestinal mucosa. The present study provides a clue to conduct a research for cancer preventive roles of GGA in a number of spices.