Development of a novel approach, the epigenome-based outlier approach, to identify tumor-suppressor genes silenced by aberrant DNA methylation

Identification of tumor-suppressor genes (TSGs) silenced by aberrant methylation of promoter CpG islands (CGIs) is important, but hampered by a large number of genes methylated as passengers of carcinogenesis. To overcome this issue, we here took advantage of the fact that the vast majority of genes methylated in cancers lack, in normal cells, RNA polymerase II (Pol II) and have trimethylation of histone H3 lysine 27 (H3K27me3) in their promoter CGIs. First, we demonstrated that three of six known TSGs in breast cancer and two of three in colon cancer had Pol II and lacked H3K27me3 in normal cells, being outliers to the general rule. BRCA1, HOXA5, MLH1, and RASSF1A had high Pol II, but were expressed only at low levels in normal cells, and were unlikely to be identified as outliers by their expression statuses in normal cells. Then, using epigenome statuses (Pol II binding and H3K27me3) in normal cells, we made a genome-wide search for outliers in breast cancers, and identified 14 outlier promoter CGIs. Among these, DZIP1, FBN2, HOXA5, and HOXC9 were confirmed to be methylated in primary breast cancer samples. Knockdown of DZIP1 in breast cancer cell lines led to increases of their growth, suggesting it to be a novel TSG. The outliers based on their epigenome statuses contained unique TSGs, including DZIP1, compared with those identified by the expression microarray data. These results showed that the epigenome-based outlier approach is capable of identifying a different set of TSGs, compared to the expression-based outlier approach.     

Early-stage formation of an epigenetic field defect in a mouse colitis model, and non-essential roles of T- and B-cells in DNA methylation induction

Epigenetic fields for cancerization are involved in development of human cancers, especially those associated with inflammation and multiple occurrences. However, it is still unclear when such field defects are formed and what component of inflammation is involved in induction of aberrant DNA methylation. Here, in a mouse colitis model induced by dextran sulfate sodium (DSS), we identified three CpG islands specifically methylated in colonic epithelial cells exposed to colitis. Their methylation levels started to increase as early as 8 weeks after DSS treatment and continued to increase until colon cancers developed at 15 weeks. In contrast to the temporal profile of DNA methylation levels, infiltration of inflammatory cells spiked immediately after the DSS treatment and then gradually decreased. Exposure of cultured colonic epithelial cells to DSS did not induce DNA methylation and it was indicated that inflammation triggered by the DSS treatment was responsible for methylation induction. To clarify components of inflammation involved, severe combined immunodeficiency (SCID) mice that lack functional T- and B-cells were similarly treated. Even in SCID mice, DNA methylation, along with colon tumors, were induced at the same levels as in their background strain of mice (C.B17). Comparative analysis of inflammation-related genes showed that Ifng, Il1b and Nos2 had expression concordant with methylation induction whereas Il2, Il6, Il10, Tnf did not. These results showed that an epigenetic field defect is formed at early stages of colitis-associated carcinogenesis and that functional T and B cells are non-essential for the formation.     

D-beta-hydroxybutyrate protects dopaminergic SH-SY5Y cells in a rotenone model of Parkinson's disease

It has been postulated that the pathogenesis of Parkinson's disease (PD) is associated with mitochondrial dysfunction. Rotenone, an inhibitor of mitochondrial complex I, provides models of PD both in vivo and in vitro. We investigated the neuroprotective effect of D-beta-hydroxybutyrate (bHB), a ketone body, against rotenone toxicity by using SH-SY5Y dopaminergic neuroblastoma cells. SH-SY5Y cells, differentiated by all-trans-retinoic acid, were exposed to rotenone at concentrations ranging from 0 to 1,000 nM. We evaluated cellular oxidation reduction by the alamarBlue assay, viability by lactate dehydrogenase (LDH) assay, and survival/death ratio by live/dead assays. Exposure to rotenone for 48 hr oxidized cells and decreased their viability and survival rate in a concentration-dependent manner. Pretreatment of cells with 8 mM bHB provided significant protection to SH-SY5Y cells. Whereas rotenone caused the loss of mitochondrial membrane potential, released cytochrome c into the cytosol, and reduced cytochrome c content in mitochondria, addition of bHB blocked this toxic effect. bHB also attenuated the rotenone-induced activation of caspase-9 and caspase-3. Administration of 0-10 mM 3-nitropropionic acid, a complex II inhibitor, also decreased the reducing power of SH-SY5Y cells measured by alamarBlue assay. Pretreatment with 8 mM bHB attenuated the decrease of alamarBlue fluorescence. These data demonstrated that bHB had a neuroprotective effect that supported the mitochondrial respiration system by reversing the inhibition of complex I or II. Ketone bodies, the alternative energy source in the mammalian brain, appear to have therapeutic potential in PD.     

Isolation and characterization of 13 microsatellite loci in the nine-spined stickleback (Pungitius pungitius) and cross-species amplification in 5 stickleback species (family Gasterosteidae)

A microsatellite-enriched genomic library was obtained from the nine-spined stickleback, the freshwater type of Pungitius pungitius, and 13 dinucleotide markers were successfully isolated and characterized. Cross-species amplification of these markers in three Pungitius species and two Gasterosteus species (family Gasterosteidae) was performed under standard conditions. The mean observed and expected heterozygosity was 0.500–0.688 and 0.508–0.750, respectively. Low allelic diversity was noted among the microsatellite loci in P. tymensis. These results confirm the genetic vulnerability of Pungitius and the need for its conservation. The newly established microsatellite markers will facilitate population-genetic and ecological studies for the conservation of sympatric sticklebacks.     

Risk factors of near-fatal deliberate self-harm behavior in self-cutting patients: a three-year follow-up study at a psychiatric clinic

Non-fatal self-injurious behavior such as cutting oneself is often performed without suicidal intent to cope with emotional distress, although it is well-known to have a close association with future suicidal behavior. However, it is unclear what kinds of clinical features are presented by such self-injuring patients with a higher suicidal tendency. In the present study, we conducted a three-year follow-up study of female self-injuring patients to examine the risk factors of "near-fatal" deliberate self-harm behavior (DSH). The subjects were 81 female outpatients who had cut themselves at least once, and who had consulted a psychiatric clinic from June 2004 to July 2004. Initial assessments included traumatic life events, clinical features of self-cutting, histories of self-poisoning, alcohol abuse (Alcohol Use Disorders Identification Test: AUDIT), impulsivity (Barratt Impulsiveness Scale, 11th version: BIS-11), symptoms of bulimia nervosa (Bulimia Investigatory Test, Edinburgh: BITE), dissociation (Adolescent Dissociative Experience Scale: ADES), Global Assessment of Functioning (GAF) score, and axis I diagnosis of DSM-IV (Diagnostic and Statistical Manual, 4th version). After three years, we investigated whether the subjects had committed fatal DSH during the follow-up term. We obtained information on fatal DSH from 67 subjects during the follow-up term. Fifteen of the 67 (22.4%) had committed near-fatal DSH at least once, and one subject committed suicide by fatal DSH. Monovariate analysis revealed that in the initial assessment, the subjects with near-fatal DSH episodes more frequently reported a history of victimization by rape in adulthood and a history of OTC (over-the-counter) drug self-poisoning, and had higher scores on the BITE and AUDIT than those without near-fatal DSH episodes. Further, multivariate analysis demonstrated that only the BITE score was a significant factor in predicting future near-fatal DSH. In conclusion, symptoms of bulimia nervosa may have important clinical implications. The BITE may be a useful tool to assess future suicidal behavior in female self-cutting patients.     

Prevalence of and risk factors for suicide-related outcomes in the World Health Organization World Mental Health Surveys Japan

Aim:  Suicide is a major public health concern in Japan but little is known about the prevalence of and risk factors for suicidal ideation, plans, and attempts. The aim of the present study was to clarify the prevalence of and risk factors for important suicide-related outcomes.
Methods:  Important suicide-related outcomes and risk factors were assessed in face-to-face interviews with 2436 adult respondents in seven areas as part of the World Health Organization (WHO) World Mental Health Survey Initiative. Mental disorders were assessed with the WHO Composite International Diagnostic Interview (CIDI).
Results:   The lifetime prevalence estimates of suicidal ideation, plans, and attempts were 10.9%, 2.1%, and 1.9%, respectively. Risk of suicide plans and attempts was highest when suicidal ideation occurred at an early age and within the first year of ideation. In middle-aged individuals, the period after first employment and the presence of mental disorders were risk factors.
Conclusions:  Risk of suicide plans and attempts is highest when suicidal ideation occurred at an earlier age and within the first year of ideation. Mental disorders are as predictive of the suicide-related outcomes examined here, and comorbidity is an important predictor.     

Prophylactic fresh frozen plasma may prevent development of hepatic VOD after stem cell transplantation via ADAMTS13-mediated restoration of von Willebrand factor plasma levels

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high approximately intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).     

Preoperative cellulose porous beads for therapeutic embolization of meningioma: provocation test and technical considerations

Introduction Cellulose porous beads (CPBs) are exceptionally uniform in size and nonabsorbable and they provide highly effective tumor devascularization. The risk of cranial nerve palsy must not be overlooked when embolization with CPBs is considered in meningioma patients. We attempted to identify patients at risk of cranial nerve palsy after meningioma embolization. Methods Prior to preoperative superselective embolization with 200 μm diameter CPBs, 141 patients with meningioma underwent provocation test with lidocaine and amytal. They were divided into two groups on the basis of whether they were or were not considered eligible for embolization. We evaluated the differences between the two groups with respect to tumor anatomy, angiographic findings, and clinical presentation and recorded complications associated with the embolization of the meningioma. Results Of the 141 patients, 128 underwent CPB embolization (group 2); 13 were not embolized because their provocation test results were positive (group 1, n = 11) or because they showed vasospasm (n = 2). Group 1 patients had meningioma in the cavernous sinus or petroclival region. Characteristically, the feeders were of middle meningeal artery origin and exhibited a posteromedial course toward the petrous apex or cavernous sinus. In group 2 patients the middle meningeal artery was the feeder, but it lacked branches coursing posteromedially. Three of these patients experienced complications which included intratumoral hemorrhage (n = 2) and post-embolization hearing disturbance (n = 1). Conclusion Patients with meningioma whose tumor-feeding arteries run posteromedially toward the petrous apex or cavernous sinus are at increased risk of post-embolization cranial nerve palsy. Appropriate protocols, including lidocaine and amytal provocation tests, may reduce the risk of complications after CPB embolization of the external carotid territory in this group of patients.