Junko Kimura

Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk.

Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (associated with amino acid changes) SNPs identified were assessed for associations with lung cancer risk by a case‐control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest association with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double‐strand break repair protein, also showed associations (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, respectively). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was associated with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages < 61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, respectively). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.

An Attenuated LC16m8 Smallpox Vaccine: Analysis of Full-Genome Sequence and Induction of Immune Protection

ABSTRACT The potential threat of smallpox bioterrorism has made urgent the development of lower-virulence vaccinia virus vaccines. An attenuated LC16m8 (m8) vaccine was developed in 1975 from the Lister strain used in the World Health Organization smallpox eradication program but was not used against endemic smallpox. Today, no vaccines can be tested with variola virus for efficacy in humans, and the mechanisms of immune protection against the major intracellular mature virion (IMV) and minor extracellular enveloped virion (EEV) populations of poxviruses are poorly understood. Here, we determined the full-genome sequences of the m8, parental LC16mO (mO), and grandparental Lister (LO) strains and analyzed their evolutionary relationships. Sequence data and PCR analysis indicated that m8 was a progeny of LO and that m8 preserved almost all of the open reading frames of vaccinia virus except for the disrupted EEV envelope gene B5R. In accordance with this genomic background, m8 induced 100% protection against a highly pathogenic vaccinia WR virus in mice by a single vaccination, despite the lack of anti-B5R and anti-EEV antibodies. The immunogenicity and priming efficacy with the m8 vaccine consisting mainly of IMV were as high as those with the intact-EEV parental mO and grandparental LO vaccines. Thus, mice vaccinated with 107 PFU of m8 produced low levels of anti-B5R antibodies after WR challenge, probably because of quick clearance of B5R-expressing WR EEV by strong immunity induced by the vaccination. These results suggest that priming with m8 IMV provides efficient protection despite undetectable levels of immunity against EEV.

Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines - 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells - were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and suppressions of oxidative stress and cell proliferation.

Nobiletin, a flavone from Citrus depressa, induces gene expression and increases the protein level and activity of neprilysin in SK-N-SH cells

Neprilysin (NEP) is one of the candidate amyloid β protein (Aβ) degrading enzymes affecting brain Aβ clearance. This enzyme declines in the brain with age, which leads to the increased Aβ deposition in Alzheimers disease (AD). Pharmacological activation of NEP during the aging process, therefore, represents a potential strategy to prevent the development of AD. To examine the influence of nobiletin on neprilysin activity, we measured cellular NEP activity in SK-N-SH cells. Moreover, NEP expression was examined by using reverse transcription - polymerase chain reaction and Western blotting. Measurement of cellular NEP activity showed that nobiletin stimulated this in a dose- and time-dependent manner in SK-N-SH cells. Moreover, nobiletin increased the expression of NEP mRNA, and then the levels of NEP protein, also in a dose- and time-dependent manner. Our findings showed that nobiletin promoted NEP gene and protein expression, resulting in enhancement of cellular NEP activity in SK-N-SH cells. This compound could be a novel Aβ-degrading compound for use in the development of disease-modifying drugs to prevent and (or) cure AD.

Nobiletin Reduces Intracellular and Extracellular β-Amyloid in iPS Cell-Derived Alzheimer's Disease Model Neurons

Alzheimers disease (AD) is the most common cause of dementia, with progressive memory impairment. Recently, neprilysin, a β-amyloid (Aβ)-degrading enzyme has become featured as a drug target for AD. Previously, we identified nobiletin from citrus peels as a natural compound possessing anti-dementia activity. In addition, we demonstrated that nobiletin improved memory in memory-impaired animals and, further, that Aβ levels were markedly decreased in the brains of these animals. We demonstrated in vitro that nobiletin up-regulates neprilysin expression and activity in human neuroblastoma cells. However, the action of nobiletin with regard to Aβ degradation under in vitro AD pathological conditions remains unclear. In this study, we examined whether nobiletin could enhance the degradation of intra- and extracellular Aβ using human induced pluripotent stem cell-derived AD model neurons, which generate an excess of Aβ1-42 due to the familial AD presenilin-1 mutation. The neurons were treated in the presence or absence of nobiletin. The results of real-time quantitative RT-PCR indicated that neprilysin mRNA levels were significantly up-regulated by nobiletin. Furthermore, immunostaining with an anti-Aβ antibody revealed that nobiletin substantially reduced the intraneuronal content of Aβ. Interestingly, the results of Aβ1-42 immunoassays confirmed that nobiletin also significantly decreased the levels of Aβ1-42 released into the cellular medium. These results suggest that nobiletin enhanced the reduction of intra- and that extracellular Aβ levels under AD pathologic conditions, and this is associated with the up-regulation of neprilysin expression. Collectively, nobiletin appears to be a promising novel prophylactic seed drug or functional food for AD.

Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.