Reduced Tumorigenicity of Mouse ES Cells and the Augmented Anti-Tumor Therapeutic Effects under Parg Deficiency.

PolyADP-ribosylation is a post-translational modification of proteins, and poly(ADP-ribose) (PAR) polymerase (PARP) family proteins synthesize PAR using NAD as a substrate. Poly(ADP-ribose) glycohydrolase (PARG) functions as the main enzyme for the degradation of PAR. In this study, we investigated the effects of Parg deficiency on tumorigenesis and therapeutic efficacy of DNA damaging agents, using mouse ES cell-derived tumor models. To examine the effects of Parg deficiency on tumorigenesis, Parg+/+ and Parg-/- ES cells were subcutaneously injected into nude mice. The results showed that Parg deficiency delays early onset of tumorigenesis from ES cells. All the tumors were phenotypically similar to teratocarcinoma and microscopic findings indicated that differentiation spectrum was similar between the Parg genotypes. The augmented anti-tumor therapeutic effects of X-irradiation were observed under Parg deficiency. These results suggest that Parg deficiency suppresses early stages of tumorigenesis and that Parg inhibition, in combination with DNA damaging agents, may efficiently control tumor growth in particular types of germ cell tumors.

Role of P2X7 receptor in Clostridium perfringens beta-toxin-mediated cellular injury.

BACKGROUND: Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X(7) receptor, which is an ATP receptor, interacts with beta-toxin. METHODS: We tested the role of P2X(7) receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X(7) receptor was further determined using an in vivo mouse model. RESULTS: Selective P2X(7) receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X(7) receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X(7) receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y(2) or P2Y(6) had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X(7)-transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X(7) receptors in vitro and colocalized with the P2X(7) receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG. CONCLUSIONS: The results of this study indicate that the P2X(7) receptor plays a role in beta-toxin-mediated cellular injury. GENERAL SIGNIFICANCE: P2X(7) receptor is a potential target for the treatment of C. perfringens type C infection.

Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/µm) and Fe-ion irradiation (200 keV/µm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/µm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.

Radiosensitization effect of poly(ADP-ribose) polymerase inhibition in cells exposed to low and high liner energy transfer radiation.

Poly(ADP-ribose) polymerase (PARP)-1 promotes base excision repair and DNA strand break repair. Inhibitors of PARP enhance the cytotoxic effects of γ-irradiation and X-irradiation. We investigated the impact of PARP inhibition on the responses to γ-irradiation (low liner energy transfer [LET] radiation) and carbon-ion irradiation (high LET radiation) in the human pancreatic cancer cell line MIA PaCa-2. Cell survival was assessed by colony formation assay after combination treatment with the PARP inhibitor AZD2281 and single fraction γ-irradiation and carbon-ion irradiation (13 and 70 keV/µm [LET 13 and LET 70]). The DNA damage response (DDR) was assessed by pulse field gel electrophoresis, western blotting and flow cytometry. Treatment with a PARP inhibitor enhanced the cytotoxic effect of γ-irradiation and LET 13 and LET 70 carbon-ion irradiation. Moreover, the radiosensitization effect was greater for LET 70 than for LET 13 irradiation. Prolonged and increased levels of γ-H2AX were observed both after γ-irradiation and carbon-ion irradiation in the presence of the PARP inhibitor. Enhanced level of phosphorylated-p53 (Ser-15) was observed after γ-irradiation but not after carbon-ion irradiation. PARP inhibitor treatment induced S phase arrest and enhanced subsequent G2/M arrest both after γ-irradiation and carbon-ion irradiation. These results suggest that the induction of S phase arrest through an enhanced DDR and a local delay in DNA double strand break processing by PARP inhibition caused sensitization to γ-irradiation and carbon-ion irradiation. Taken together, PARP inhibitors might be applicable to a wide therapeutic range of LET radiation through their effects on the DDR.

PPARalpha is involved in photoentrainment of the circadian clock.

We found that bezafibrate, a ligand of peroxisome proliferator-activated receptor alpha (PPARalpha), advances the active phase of mice under light-dark (LD) conditions in a photoperiod-dependent manner. Bezafibrate gradually advanced the activity onset that consequently almost completely reversed the active phase from the dark to the light period under a long photoperiod (18 h of light and 6 h of darkness: LD 18 : 6). The activity onset was not changed under a short photoperiod (LD 8 : 16) or under constant illumination. These observations suggest that PPARalpha is involved in entrainment of the circadian clock to environmental LD conditions.

Role of Ca2+-binding motif in cytotoxicity induced by Clostridium perfringens iota-toxin.

Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). We investigated the role of the conserved Ca(2+)-binding motif of Ib in the cytotoxicity of iota-toxin. The cytotoxicity of iota-toxin increased with an increase in the concentration of extracellular Ca(2+). A surface plasmon resonance analysis showed that the binding of Ia to the oligomer of Ib is dependent on the concentration of Ca(2+). However, the addition of Ca(2+) had no effect on the binding of (125)I-labeled Ib to the cells. We replaced Asp-8, -10, and -12 in the Ca(2+)-binding motif of Ib with alanine. D8A, D10A, and D12A bound to the cell and formed an oligomer at about half of the wild-type Ib. The cytotoxicity of Ib variants in the presence of Ia was about 500-fold less than that of wild-type Ib. Immunofluorescence study showed that these variants were internalized in the early endosomes like wild-type Ib. However, wild-type Ib-induced internalization of Ia in the cells, but these variants did not. The result indicates that the conserved Ca(2+)-binding motif in the N-terminal region of Ib plays a role in the interaction of Ib with Ia in the presence of Ca(2+).

Circadian variations in coagulation and fibrinolytic factors among four different strains of mice.

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI-1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

PPARalpha is a potential therapeutic target of drugs to treat circadian rhythm sleep disorders.

Recent progress at the molecular level has revealed that nuclear receptors play an important role in the generation of mammalian circadian rhythms. To examine whether peroxisome proliferator-activated receptor alpha (PPARalpha) is involved in the regulation of circadian behavioral rhythms in mammals, we evaluated the locomotor activity of mice administered with the hypolipidemic PPARalpha ligand, bezafibrate. Circadian locomotor activity was phase-advanced about 3h in mice given bezafibrate under light-dark (LD) conditions. Transfer from LD to constant darkness did not change the onset of activity in these mice, suggesting that bezafibrate advanced the phase of the endogenous clock. Surprisingly, bezafibrate also advanced the phase in mice with lesions of the suprachiasmatic nucleus (SCN; the central clock in mammals). The circadian expression of clock genes such as period2, BMAL1, and Rev-erbalpha was also phase-advanced in various tissues (cortex, liver, and fat) without affecting the SCN. Bezafibrate also phase-advanced the activity phase that is delayed in model mice with delayed sleep phase syndrome (DSPS) due to a Clock gene mutation. Our results indicated that PPARalpha is involved in circadian clock control independently of the SCN and that PPARalpha could be a potent target of drugs to treat circadian rhythm sleep disorders including DSPS.

Bidirectional CLOCK/BMAL1-dependent circadian gene regulation by retinoic acid in vitro.

A central circadian clock located in the suprachiasmatic nucleus (SCN) of the mammalian hypothalamus entrains peripheral clocks through both neural and humoral factors. Although candidates for entrainment factors have been described, their details remain obscure. Here, we screened ligands for nuclear receptors that affect CLOCK/BMAL1-dependent transactivation of the mouse Period1 (mPer1) gene in NIH3T3 cells. We found that retinoic acids (RAs) significantly up-regulate mPer1 expression in an E-box-dependent manner. We also found that RAs up-regulate the expression of other E-box-dependent circadian genes such as mPer2, arginine vasopressin (mAVP), and peroxisome proliferator-activated receptor alpha (mPPARalpha). Surprisingly, the effect of RAs on CLOCK/BMAL1 (E-box)-dependent mRNA expression was bidirectional and depended on the presence of exogenous retinoic acid receptor alpha (RARalpha). These results suggest that RAs regulate the CLOCK/BMAL1-dependent transcription of circadian genes in a complex manner.

Circadian expression of clock genes is maintained in the liver of Vitamin A-deficient mice.

In mammals, circadian oscillators exist not only in the central clock of the suprachiasmatic nucleus (SCN) but also in peripheral tissues such as the liver, heart and kidneys. Peripheral clocks are entrained to the SCN clock by both neural and humoral signals. Vitamin A might be one candidate that synchronizes peripheral clocks by activating its ligand-dependent nuclear receptors in mammals. The present study examines the effect of a Vitamin A deficiency on the circadian expression of clock genes in the mouse liver. Serum Vitamin A levels remained constant throughout the day in control mice, and were significantly reduced in Vitamin A-deficient mice. Northern blots showed that circadian expression of the clock genes mPer1, mPer2, Clock, and BMAL1, and of the clock-controlled output gene D-site binding protein (DBP), was maintained in Vitamin A-deficient mice. Our results suggest that dietary Vitamin A is not essential for generating circadian rhythms of peripheral clocks in mammals.

Genome-wide expression analysis reveals 100 adrenal gland-dependent circadian genes in the mouse liver.

Recent progress in genome-wide expression analysis has identified hundreds of circadian genes not only in the suprachiasmatic nucleus (the mammalian master clock) but also in peripheral tissues, such as heart, liver and kidney of mammals. Glucocorticoid is thought to be a circadian time cue for mammalian peripheral clocks. To identify the genes of which the circadian expression is regulated by endogenous glucocorticoids, we performed DNA microarray analysis using hepatic RNA from adrenalectomized (ADX) and sham-operated mice. We identified 169 genes that fluctuated between day and night in the livers of the sham-operated mice. Among these, 100 lost circadian rhythmicity in ADX mice. These included the genes for key enzymes of liver metabolic functions, such as glucokinase, HMG-CoA reductase and glucose-6-phosphatase. The circadian expression of Lpin1, FKBP51 and S-adenosyl methionine decarboxylase was also abolished in the ADX mice. On the other hand, although the circadian expression of clock or clock-related genes, such as mPer2, DBP, E4BP4, mDec1, Usp2 and Wee1 remained almost totally intact in the liver of ADX mice, it was extremely damped in homozygous Clock mutant mice. The present findings suggested that one type of hepatic circadian genes in mice is transcriptionally regulated by core components of the circadian clock, such as CLOCK and BMAL1, and that the other depends on the adrenal gland.

Differential circadian expression of endothelin-1 mRNA in the rat suprachiasmatic nucleus and peripheral tissues.

The vasoconstrictor endothelin-1 (ET-1) is implicated in normal neuronal functions. Here we show the circadian expression of ET-1 mRNA in the rat suprachiasmatic nucleus (SCN) that is considered to be the location of the central circadian pacemaker, as well as in peripheral tissues including the brain, heart, and lungs. The expression of ET-1 in the SCN oscillated with a peak at Zeitgeber time (ZT) 4 under light-dark conditions. A significant number of cells in the SCN was stained with ET-1 probe during circadian time (CT) 6, but there was no significant staining at CT18 by mRNA in situ hybridization. The circadian rhythm of ET-1 mRNA in the whole brain also oscillated, but peaked at ZT20. Endothelin-1 expression in the lungs and heart peaked at ZT12 and ZT20, respectively. The results are the first description of the circadian expression of ET-1 mRNA. The diversity of rhythmic expressions among the SCN, whole brain, lungs and heart suggests that ET-1 has different functions in these tissues.

CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor alpha (PPARalpha) in mice.

PPARalpha (peroxisome-proliferator-activated receptor alpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. In the present study, we show that circadian expression of mouse PPARalpha mRNA requires the basic helix-loop-helix PAS (Per-Arnt-Sim) protein CLOCK, a core component of the negative-feedback loop that drives circadian oscillators in mammals. The circadian expression of PPARalpha mRNA was abolished in the liver of homozygous Clock mutant mice. Using wild-type and Clock-deficient fibroblasts derived from homozygous Clock mutant mice, we showed that the circadian expression of PPARalpha mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner. Transient transfection and EMSAs (electrophoretic mobility-shift assays) revealed that the CLOCK-BMAL1 (brain and muscle Arnt-like protein 1) heterodimer transactivates the PPARalpha gene via an E-box-rich region located in the second intron. This region contained two perfect E-boxes and four E-box-like motifs within 90 bases. ChIP (chromatin immunoprecipitation) also showed that CLOCK associates with this E-box-rich region in vivo. Circadian expression of PPARalpha mRNA was intact in the liver of insulin-dependent diabetic and of adrenalectomized mice, suggesting that endogenous insulin and glucocorticoids are not essential for the rhythmic expression of the PPARalpha gene. These results suggested that CLOCK plays an important role in lipid homoeostasis by regulating the transcription of a key protein, PPARalpha.

Tissue-specific augmentation of circadian PAI-1 expression in mice with streptozotocin-induced diabetes.

Diabetes is associated with an excess risk of cardiac events, and the risk for infarction is partly determined by plasminogen activator inhibitor-1 (PAI-1). We found that plasma total and active PAI-1 levels increased in a circadian manner in mice with streptozotocin (STZ)-induced diabetes. Circadian expression of PAI-1 mRNA in the lung, heart, liver, and kidney increased in a tissue-specific manner. Peak to peak comparisons revealed that the mRNA expression levels increased by 1.7, 1.7, 1.2, and 1.6-fold in the heart, lung, liver, and kidney, respectively. In contrast, the circadian expression of the clock gene, mPer2, was preserved in the diabetic mice, suggesting that the altered expression of PAI-1 mRNA did not arise due to impaired circadian clocks. Our results suggest that impairment of the coagulation and fibrinolytic systems induced by diabetes is partly due to impaired circadian PAI-1 fluctuation at the level of mRNA expression.