Defective DNA Strand Break Repair Causes Chromosomal Instability and Accelerates Liver Carcinogenesis in Mice

Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. Conclusion: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC

A random mutation capture assay to detect genomic point mutations in mouse tissue

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo

Targeting cap-dependent translation blocks converging survival signals by AKT and PIM kinases in lymphoma

PIM kinase expression in human lymphomas can influence the outcome of chemotherapy, and blocking cap-dependent translation can reverse PIM-mediated rapamycin resistance in murine lymphomas

Identification of KIF3A as a Novel Candidate Gene for Childhood Asthma Using RNA Expression and Population Allelic Frequencies Differences

Asthma is a chronic inflammatory disease with a strong genetic predisposition. A major challenge for candidate gene association studies in asthma is the selection of biologically relevant genes.Using epithelial RNA expression arrays, HapMap allele frequency variation, and the literature, we identified six possible candidate susceptibility genes for childhood asthma including ADCY2, DNAH5, KIF3A, PDE4B, PLAU, SPRR2B. To evaluate these genes, we compared the genotypes of 194 predominantly tagging SNPs in 790 asthmatic, allergic and non-allergic children. We found that SNPs in all six genes were nominally associated with asthma (p<0.05) in our discovery cohort and in three independent cohorts at either the SNP or gene level (p<0.05). Further, we determined that our selection approach was superior to random selection of genes either differentially expressed in asthmatics compared to controls (p = 0.0049) or selected based on the literature alone (p = 0.0049), substantiating the validity of our gene selection approach. Importantly, we observed that 7 of 9 SNPs in the KIF3A gene more than doubled the odds of asthma (OR = 2.3, p<0.0001) and increased the odds of allergic disease (OR = 1.8, p<0.008). Our data indicate that KIF3A rs7737031 (T-allele) has an asthma population attributable risk of 18.5%. The association between KIF3A rs7737031 and asthma was validated in 3 independent populations, further substantiating the validity of our gene selection approach.Our study demonstrates that KIF3A, a member of the kinesin superfamily of microtubule associated motors that are important in the transport of protein complexes within cilia, is a novel candidate gene for childhood asthma. Polymorphisms in KIF3A may in part be responsible for poor mucus and/or allergen clearance from the airways. Furthermore, our study provides a promising framework for the identification and evaluation of novel candidate susceptibility genes

Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases

Three Novel Downstream Promoter Elements Regulate MHC Class I Promoter Activity in Mammalian Cells

BACKGROUND: MHC CLASS I TRANSCRIPTION IS REGULATED BY TWO DISTINCT TYPES OF REGULATORY PATHWAYS: 1) tissue-specific pathways that establish constitutive levels of expression within a given tissue and 2) dynamically modulated pathways that increase or decrease expression within that tissue in response to hormonal or cytokine mediated stimuli. These sets of pathways target distinct upstream regulatory elements, have distinct basal transcription factor requirements, and utilize discrete sets of transcription start sites within an extended core promoter. METHODOLOGY/PRINCIPAL FINDINGS: We studied regulatory elements within the MHC class I promoter by cellular transfection and in vitro transcription assays in HeLa, HeLa/CIITA, and tsBN462 of various promoter constructs. We have identified three novel MHC class I regulatory elements (GLE, DPE-L1 and DPE-L2), located downstream of the major transcription start sites, that contribute to the regulation of both constitutive and activated MHC class I expression. These elements located at the 3' end of the core promoter preferentially regulate the multiple transcription start sites clustered at the 5' end of the core promoter. CONCLUSIONS/SIGNIFICANCE: Three novel downstream elements (GLE, DPE-L1, DPE-L2), located between +1 and +32 bp, regulate both constitutive and activated MHC class I gene expression by selectively increasing usage of transcription start sites clustered at the 5' end of the core promoter upstream of +1 bp. Results indicate that the downstream elements preferentially regulate TAF1-dependent, relative to TAF1-independent, transcription

A Community-Based Oral Health Promotion Program for Migrant Older Adults

A study was designed to assess the oral health of older Greek and Italian adults attending community clubs and living in Melbourne, Australia. The study also aimed to evaluate a community-based health promotion intervention on the oral health knowledge, attitude and practices of older Greeks/Italians. This paper discusses the design of an oral health promotion program and presents its qualitative and quantitative evaluation. Methods: The study included some 800 older adults who completed an oral health interview in their native language and underwent a detailed dental examination. The oral health promotion intervention consisted of three components: i) a series of oral health seminars held at the clubs in the participants' native languages, ii) the provision of oral care products relevant to each oral health seminar and iii) the production of oral health information sheets. The intervention program was known as the ORHIS (Oral Health Information Seminars/Sheets). Results: A total of 520 older adults participated in the evaluation. At post-test, the experimental groups were significantly more likely than the control groups to have improved oral health attitudes, oral health knowledge, self-assessed physical health status, and dentures hygiene. Positive outcomes were also achieved for self-reported oral hygiene practices and use of oral health services, but not for any of the clinical variables. Conclusion: The qualitative evaluation indicated that the oral health promotion program was effectively delivered in ethnic social clubs, highly acceptable to these communities and valued by participants in the study.Implications of this evaluation for the design and implementation of oral health interventions in migrant older adults groups and clubs are discussed and a number of recommendations made

Self-reported and clinically determined oral health status predictors for quality of life in dentate older migrant adults

Objective: This paper reports the impact of oral health on the quality of life (QOL) of Southern European, dentate older adults, living independently in Melbourne, Australia. Participants were recruited through ethnic social clubs and interviewed about oral health, general health, sociodemographics, and QOL using the Medical Outcomes Study Short-Form 12 (SF-12). The SF-12's physical and mental health component summary scores (PCS and MCS, respectively) were computed. The Oral Health Impact Profile (OHIP-14) assessed the specific impact of oral health on QOL. Participants were also given a clinical oral examination. Results: A total of 603 eligible older adults volunteered; 308 were from Greek background and 295 were from Italian background. Mean age was 67.7 years (SD 6.2), with 63.7% being female. The PCS score had a mean value of 45.8 (SD 11.8), and MCS had a mean of 47.8(SD 5.7). PCS was associated with, periodontal status, chronic health condition, self-perceived oral health needs, self-assessed oral health status, oral health impact score and the interaction between gender and level of education [F(11 552) ¼ 10.57;