哺乳類概日リズムにおける新規時計遺伝子CHRONOの機能解析

広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

Unravelling disparate roles of NOTCH in bladder cancer.

The Notch pathway has been implicated in both oncogenic and tumour-suppressive roles in cancer depending on the tissue type and cellular context. However, until recently, little was known about the pathway in bladder cancer. Studies have revealed that NOTCH1 copy number and expression are decreased in bladder cancer and NOTCH1 activation in bladder cancer cell lines reduces proliferation, suggesting that NOTCH1 acts as a tumour suppressor. Furthermore, in transgenic models, bladder cancer is promoted by bladder-specific inactivation of a component of the γ-secretase complex, which liberates the intracellular domain of neurogenic locus Notch homologue protein (NOTCH) and starts the signalling cascade. By contrast, further work has demonstrated that NOTCH2 acts as an oncogene that promotes cell proliferation and metastasis through epithelial-to-mesenchymal transition, cell cycle progression, and maintenance of stemness. Studies indicating that NOTCH1 and NOTCH2 have opposite effects on the progression of bladder cancer could give rise to potential therapeutic approaches aimed at blocking or restoring the Notch pathway

A novel protein, CHRONO, functions as a core component of the mammalian circadian clock.

Circadian rhythms are controlled by a system of negative and positive genetic feedback loops composed of clock genes. Although many genes have been implicated in these feedback loops, it is unclear whether our current list of clock genes is exhaustive. We have recently identified Chrono as a robustly cycling transcript through genome-wide profiling of BMAL1 binding on the E-box. Here, we explore the role of Chrono in cellular timekeeping. Remarkably, endogenous CHRONO occupancy around E-boxes shows a circadian oscillation antiphasic to BMAL1. Overexpression of Chrono leads to suppression of BMAL1-CLOCK activity in a histone deacetylase (HDAC) -dependent manner. In vivo loss-of-function studies of Chrono including Avp neuron-specific knockout (KO) mice display a longer circadian period of locomotor activity. Chrono KO also alters the expression of core clock genes and impairs the response of the circadian clock to stress. CHRONO forms a complex with the glucocorticoid receptor and mediates glucocorticoid response. Our comprehensive study spotlights a previously unrecognized clock component of an unsuspected negative circadian feedback loop that is independent of another negative regulator, Cry2, and that integrates behavioral stress and epigenetic control for efficient metabolic integration of the clock

Six-year Experience of Permanent Prostate Brachytherapy for Clinically Localized Prostate Cancer

This report presents the outcome of prostate permanent brachytherapy (PPB). One hundred and seventy-two patients with clinically localized prostate cancer were treated with permanent brachytherapy using iodine-125 seeds (125-I) at Hiroshima University Hospital from July 2004 to June 2010. This study evaluated the efficacy of PPB in these patients. The median patient age was 69 years (range 53 to 82 years), the median prostate-specific antigen (PSA) value before biopsy was 6.75 ng/ml (range 3.5 to 47.9 ng/ml), and the median prostate volume was 23.1 ml (range 10.1 to 57 ml). The median follow-up was 37 months (range 1 to 72 months). The serum PSA levels decreased continuously after PPB throughout the entire follow-up period in 97% of patients without neoadjuvant hormonal therapy. No relapse occurred during the follow-up period in patients at low risk. Our 6-year experience suggests that PPB is effective for localized prostate cancer. Patients with prostate cancer that does not require combined External beam radiation therapy (EBRT) have the best chance of responding to treatment

CHRONO.

<p>(A) Cell-free synthesis of CHRONO (GM129). Autoradiogram of <i>in vitro</i> translated CHRONO, CHRONO–FLAG, and luciferase (control). Protein was synthesized in the presence of [³⁵S]methionine and resolved on a 10% SDS-PAGE gel. (B) Temporal mRNA expression of <i>Chrono</i> (solid lines with black circles) and <i>Bmal1</i> (dotted lines with white circles) in mouse peripheral tissues. The abscissa represents time in CT and the ordinate the mRNA amounts. The relative levels of mRNA were normalized to the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. The maximum mRNA amount was set to 100. Plots and error bars represent mean ± S.E.M of triplicate samples. (C) BMAL1 ChIP-seq tag enrichments in the whole brain sample were located on the <i>Chrono</i> promoter region in the UCSC genome browser view. (D) The effects of overexpression of BMAL1 and CLOCK (B/C) on the <i>Chrono</i> promoter modification were evaluated by using a luciferase assay. The left scheme shows each construct on a black line indicating the position of the E-box element and a red line indicating the TSS. The basal transcriptional activity of each <i>Chrono</i> promoter was set to 1. Horizontal bars represent means ± S.E.M. of four samples (*<i>p</i><0.005, **<i>p</i><0.0005, Student's <i>t</i> test). (E) E-boxes of the <i>Chrono</i> promoter required for transcriptional oscillation. The cell-culture-based bioluminescent reporter assay was performed with the each construct in (D). The abscissa indicates the day in culture, and the ordinate the relative bioluminescence intensity (kcpm, 1,000 photon counts per minute). Shaded area indicates ± S.E.M of four samples.</p

<i>Chrono</i> in the <i>Avp</i> neurons plays a central role in behavioral rhythms.

<p>(A) The targeting strategy is illustrated with the structure of the WT <i>Chrono</i> allele, the targeting vector, the floxed allele <i>Chrono</i>^flx. <i>Chrono</i> exons are shown as open boxes; 5′ genomic DNA, intronic sequences, and 3′ genomic DNA as solid lines. The Pr Neo pA and the Pr DT-A pA cassettes are indicated as open boxes. The loxP sequences are shown as triangles. (B) Representative actograms of <i>Chrono</i>^flx/flx and <i>Avp</i>Cre <i>Chrono</i>^flx/flx mice. Animals were maintained on 12–12 h LD cycles initially and transferred to DD. Shaded area indicates dark phase. (C) Representative chi-squared periodograms of <i>Chrono</i>^flx/flx and <i>Avp</i>Cre <i>Chrono</i>^flx/flx locomotor activities for 1 wk under DD. (D) Comparison of free-running periods estimated for <i>Chrono</i>^flx/flx and <i>Avp</i>Cre <i>Chrono</i>^flx/flx mice. Pairwise comparisons indicated that there were no significant differences in circadian periods under LD compared with the <i>Chrono</i>^flx/flx littermates. However, a significantly longer free-running period was observed in <i>Avp</i>Cre <i>Chrono</i>^flx/flx mice compared with the <i>Chrono</i>^flx/flx littermates in DD. <i>Avp</i>Cre <i>Chrono</i>^flx/flx mice (24.04±0.12 h, <i>n</i> = 6, male) and <i>Chrono</i>^flx/flx siblings (23.84±0.06 h, <i>n</i> = 8, male) in DD (mean period ± S.D.; *<i>p</i><0.01, Student's <i>t</i> test). (E) Basal locomotor activity. Amounts of activities during the light phase (left) and the dark phase (right) for 1 wk in LD show no significant differences. <i>n</i> = 8 for <i>Chrono</i>^flx/flx and <i>n</i> = 6 for <i>Avp</i>Cre <i>Chrono</i>^flx/flx.</p

CHRONO acts as a transcriptional repressor through an independent pathway.

<p>(A and B) <i>Cry2</i> KO MEFs (A) and <i>Cry2</i>sh/NIH3T3 cells (B) were transfected with indicated combinations of expression vectors. <i>Chrono</i> inhibited BMAL1–CLOCK (B/C) complex-induced transcriptional activity in the m<i>Per2</i> promoter. The effects of overexpression of BMAL1, CLOCK, CRY2, and CHRONO proteins on <i>Per2</i> transcription were evaluated by using luciferase assays. The basal transcription activity of <i>Per2</i> promoter was set to 1. Data are means ± S.E.M. of triplicate samples. **<i>p</i><0.001, Student's <i>t</i> test. (C and D) Bioluminescence rhythms (C) and periods (D) of <i>Cry2</i>sh–<i>Bmal1</i> promoter luc/NIH3T3 cells transfected with indicated expression vectors. All detrended traces (6–7 samples) from each set of experiments (Flag, <i>Cry2</i>, and <i>Chrono</i> overexpression) are superposed. The baselines are intentionally separated for each case for ease of view. The abscissa indicates the day in culture, and the ordinate the relative bioluminescence intensity (kcpm, 1,000 photon counts per minute). <i>Cry2</i>sh/NIH3T3 cells prolonged the period, and <i>Chrono</i> recovered the period length similar to <i>Cry2</i>. *<i>p</i><0.005. (E) Model prediction of <i>Bmal1</i> transcript expression in the <i>Cry2</i> knockdown (Flag, black), under additional constitutive overexpression of <i>Cry2</i> (blue), and <i>Chrono</i> (red). The baselines are separated intentionally for ease of view. (F) Raw actograms of WT, <i>Chrono</i>, <i>Cry1</i> double KO and <i>Cry1</i> KO mice and comparison of free-running period estimates. A significantly shorter free-running period was observed in <i>Chrono</i>, <i>Cry1</i> double KO and <i>Cry1</i> KO mice compared to WT in DD (G, middle), in line with the prediction through <i>in silico</i> simulation (G, right). <i>Chrono</i>, <i>Cry1</i> double KO mice did not show arrhythmicity in DD. *<i>p</i><0.05, Student's <i>t</i> test. (H) Model prediction of <i>Per2</i> mRNA time courses in <i>Cry1</i>, <i>Cry2</i> double KO (blue) and <i>Cry1</i>, <i>Cry2</i>, <i>Chrono</i> triple KO (red) compared to WT (black). The amounts of KO transcripts are set to 0 from the beginning of the simulated day 0, and dynamics before reaching steady state is illustrated.</p

Endogenous CHRONO rhythmically binds to the E-box on circadian gene promoter.

<p>(A and B) ChIP analysis for CHRONO and IgG (negative control). The CHRONO occupancies at the endogenous E-boxes of <i>Per2</i> (A) and <i>Dbp</i> (B) promoters were detected in the two different NIH3T3 cells 32 or 44 h after induction with dexamethasone. The graphs showed relative real-time PCR values compared to input. Data are means ± S.E.M. of three samples (*<i>p</i><0.05, **<i>p</i><0.01, Student's <i>t</i> test). (C) ChIP abundance of CHRONO and BMAL1 at the <i>Per2</i> promoter E-box in NIH3T3 cells after 100 nM dexamethasone stimulation. The graph showed relative real-time PCR values. The data were plotted as percentages relative to the average (1.0). Data are means ± S.E.M. of 3–4 samples.</p