Yasuhito Ohsaka

Cancer Prevention by Phytochemicals

Information has been accumulated indicating that diets rich in vegetables and fruits can reduce the risk of a number of chronic diseases, including cancer, cardiovascular disease, diabetes and age-related macular degeneration. Phytochemicals (various factors in plant foods), such as carotenoids, antioxidative vitamins, phenolic compounds, terpenoids, steroids, indoles and fibers, have been considered responsible for the risk reduction. Among them, a mixture of natural carotenoids has been studied extensively and proven to show beneficial effects on human cancer prevention.

Polymorphisms in Promoter Sequences of the p15 INK4B and PTEN Genes of Normal Japanese Individuals

Gene promoter regions of p15INK4B, a cyclin-dependent kinase inhibitor, and phosphatase and tensin homolog (PTEN), a dual-function protein and lipid phosphatase, interact with regulatory factors for gene transcription and methylation. Normal individuals exhibit sequence polymorphisms in these regulatory genes. We isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced promoter regions of the p15INK4B and PTEN genes. We also examined the influence of polymorphisms on promoter activity in several cell lines. We identified polymorphisms at positions −699, −394, and −242 and an insertion at position −320 in the p15INK4B gene and a polymorphism at position −1142 in the PTEN gene. Reporter gene analysis revealed that these polymorphisms influenced transcriptional regulation in their cell lines. Our results indicate for the first time that promoter sequences of the p15INK4B and PTEN genes differ among normal Japanese individuals and that promoter polymorphisms can influence gene transcription.

Polymorphisms in promoter sequences of MDM2, p53, and p16INK4a genes in normal Japanese individuals

Research has been conducted to identify sequence polymorphisms of gene promoter regions in patients and control subjects, including normal individuals, and to determine the influence of these polymorphisms on transcriptional regulation in cells that express wild-type or mutant p53. In this study we isolated genomic DNA from whole blood of healthy Japanese individuals and sequenced the promoter regions of the MDM2, p53, and p16INK4a genes. We identified polymorphisms comprising 3 nucleotide substitutions at exon 1 and intron 1 regions of the MDM2 gene and 1 nucleotide insertion at a poly(C) nucleotide position in the p53 gene. The Japanese individuals also exhibited p16INK4a polymorphisms at several positions, including position -191. Reporter gene analysis by using luciferase revealed that the polymorphisms of MDM2, p53, and p16INK4a differentially altered luciferase activities in several cell lines, including the Colo320DM, U251, and T98G cell lines expressing mutant p53. Our results indicate that the promoter sequences of these genes differ among normal Japanese individuals and that polymorphisms can alter gene transcription activity.

3T3-L1 adipocytes possess anandamide- and epinephrine-responsive machinery for MDM2 distribution to the plasma membrane.

The effects of biomolecules on peripheral tissues and their responsive machinery are not well understood. We examined MDM2 level in the plasma membrane (PM) and total MDM2 level of 3T3-L1 adipocytes treated with biomolecular anandamide, epinephrine, and other agents for 15 min. We also examined biomolecular responses in cells treated with mithramycin A, a binding inhibitor, or cells exposed to cooling and cell viability. Immunoblotting revealed that PM MDM2 level increased and total MDM2 level was not altered following treatment with anandamide, epinephrine, capsaicin, CL316243, and aluminum fluoride. PM MDM2 distribution caused by a biomolecular concentration was maintained by treatment with mithramycin A and exposure of cells to 28°C or 32°C but not to 18°C, and PM MDM2 levels after treatment with high concentrations of biomolecules were altered upon exposure to the inhibitor and mild hypothermia. These conditions did not decrease cell viability. Our findings indicate that 3T3-L1 adipocytes possess molecular machinery that responds differentially to anandamide and epinephrine under the inhibitor treatment and cool temperature conditions and that is sensitive to other agents (which mimic biomolecular responses); these machineries can induce subcellular alterations in molecular interactions. We provide information helpful for clarifying biomolecular responsive machinery present in 3T3-L1 adipocytes.

MDM2-related responses in 3T3-L1 adipocytes exposed to cooling and subsequent rewarming

Insulin-like growth factor-I and insulin induce the production of phospho-Ser-166 MDM2, a target of Akt, and influence the formation of the MDM2 complex. The glycolipid hormone insulin differentially activates phosphatidylinositol 3-kinase (PI3K)/Akt pathways in 3T3-L1 (L1) adipocytes incubated at 19 °C. Responses of L1 adipocytes to different temperature changes and their regulatory mechanisms are poorly understood. We exposed L1 adipocytes to cooling and subsequent rewarming in the presence or absence of wortmannin, a PI3K inhibitor, or mithramycin A, a transcription inhibitor, and examined the induction of phospho-Ser-166 MDM2 and MDM2 and the subcellular formation of the MDM2 complex using western blot analysis. Exposure to 28 and 18 °C induced phospho-MDM2 in cells and increased the level of MDM2 in the plasma membrane of cells. These temperatures did not affect the total MDM2 level. Similar results were obtained when the cells were treated with insulin. Exposure to 4 °C increased the total MDM2 level and did not induce phospho-MDM2, which was induced by rewarming at 37 °C after cooling at 4°C without any alteration in the protein level. Mithramycin A (10 μM) did not alter the increase in protein level induced at 4 °C. The induction of phospho-molecules at 28 and 18 °C was impaired slightly by 1 μM of wortmannin but not by 0.1 μM of wortmannin. This low concentration of wortmannin completely blocked the induction of phospho-MDM2 by rewarming. Our results indicate that temperature changes induce MDM2-related responses, including those that are stimulated by receptor responses and dependent on a kinase inhibitor, in L1 adipocytes.

Cooling-increased phospho-β-arrestin-1 and β-arrestin-1 expression levels in 3T3-L1 adipocytes

Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator β-arrestin-1 and phospho-Ser-412 β-arrestin-1 in 3T3-L1 adipocytes exposed to 4-37 °C or treated with some molecular agents at 37°C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5h and to 28 and 32 °C for 24 and 48 h increased phospho-β-arrestin-1 and β-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5h increased β-arrestin-1 level. Serum removal and rewarming abolished β-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24h increased the level of β-arrestin-1 but not that of phospho-β-arrestin-1. The level of phospho-β-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter β-arrestin-1 expression at 37 °C but impaired the induction of phospho-β-arrestin-1 at 28 and 32 °C without affecting the induction of β-arrestin-1. We show that cold-induced β-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for β-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for β-arrestin-1 and elucidating low-temperature responses.

Normal Japanese individuals harbor polymorphisms in the p14ARF/INK4 locus promoters and/or other gene introns. — Variation in nucleotide sequences in each individual

In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced promoters in the p14ARF/INK4 (p16INK4a and p15INK4b) locus genes and introns in murine double minute 2, vitamin D receptor, and presenilin 1 genes. We found nucleotide alterations in the promoters, including substitutions at positions −2610 and −1536 and deletions at positions −4489, −4488 to −4483, −2241 to −2240, and −2221 to −2218 in p14ARF and substitutions at positions −1643 and −1270 in p15INK4b, and we found that each individual harbored polymorphisms in the locus promoters and/or introns. Some polymorphic nucleotides were included in the same set of associatively altered nucleotides. Reporter gene analysis by using luciferase revealed that altered nucleotides, including those containing the set, changed luciferase gene activity in some cell lines. The results of this study show that multi-nucleotide sequences are altered in each normal Japanese individual.