Point mutations in normal lungs of smokers and non-smokers

Thesis (Ph. D. in Genetic Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 139-161).It is a widely-held hypothesis that environmental mutagens play an essential role in human somatic and germinal cell mutagenesis. In particular, the finding of small amounts of chemical mutagens in cigarette smoke has led to the general hypothesis that mutagens in cigarette smoke induce oncomutations and thus account for the carcinogenic effect of cigarette smoking in human lungs. However, this hypothesis has not been tested by an assay of nuclear point mutations in lungs of smokers and nonsmokers. Mismatch amplification mutation assay (MAMA), an effective form of allele-specific PCR, was applied for detection of point mutations in TP53 bp742, bp746 and bp747, K-ras bp35 and HPRT bp508 from a total of 291 tracheal-bronchial epithelial sectors from six smokers and nine non-smokers, yielding 949 individual mutational assays. The conditions of MAMA for each target point mutations were optimized such that the sensitivity of each was equal to or below 10⁻⁵. Lung epithelial sectors of 2.3x10⁶ cells in average contained 0-200 mutant cells in general, equivalent to mutant fractions (MFs) of 0-10⁻⁴ with an exception of rare sectors with MF larger than 4x 10⁻⁴ (4.6%). Noticeably, the distributions of the MFs among sectors did not vary appreciably with the donor's smoking status. The mean MFs per lung were very similar between smokers and non-smokers for all five target mutations assayed (p >> 0.05). The mean MFs were slightly higher in females than males (p = 0.015). The mean MFs increased with age of the subjects although the correlation did not reach statistical significance due to large variances within the same age group. The distributions of MF among sectors of smokers and non-smokers did not differ significantly by Kolmogorov-Smirnov test(cont.) for all target mutations but HPRT. By using hypothetical turnover unit sizes and Poisson distribution, the turnover unit size of human tracheal bronchial epithelium was estimated as 64 cells (p = 0.05). These observations do not support the widely-held hypothesis that cigarette smoking causes lung cancer through its induction of point mutations in nuclear genes. The current findings demonstrate the necessity of investigation on alternative mechanisms for tobacco smoke in lung carcinogenesis.by Hiroko Sudo.Ph.D.in Genetic Toxicolog

Radiotherapy for inoperable and refractory endometriosis presenting with massive hemorrhage: a case report

INTRODUCTION: Many patients with endometriosis are treated with medication or by surgical approaches. However, a small number of patients do not respond to medication and are inoperable because of comorbidities. This case report shows the effectiveness of radiotherapy for refractory endometriosis and includes a time series of serum estradiol levels. CASE PRESENTATION: A 47-year-old Asian woman presented to our facility with uncontrolled endometriosis refractory to medication. Our patient was considered inoperable because of severe idiopathic thrombocytopenic purpura, and underwent radiotherapy for massive genital bleeding requiring blood transfusions. A radiation dose of 20Gy in 10 fractions was delivered to the pelvis, including the bilateral ovaries, uterus, and myomas. An additional 10Gy in five fractions was delivered to the endometrium to control residual bleeding. Genital bleeding was completely inhibited on day 46 after radiotherapy. Hormonal analysis revealed that radiotherapy induced post-menopausal status. Two years after radiotherapy, atypical genital bleeding had not recurred and has been well controlled without side effects. CONCLUSIONS: Disrupted ovarian function is an adverse effect of radiotherapy. However, radiotherapy can be useful for inducing menopause. In cases of medication-refractory or inoperable endometriosis, radiotherapy would be an effective treatment option

Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102200/1/embj2009300-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102200/2/embj2009300.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102200/3/embj2009300-sup-0003.pd

Risk prediction models for dementia constructed by supervised principal component analysis using miRNA expression data

Alzheimer’s disease (AD) is the most common subtype of dementia, followed by Vascular Dementia (VaD), and Dementia with Lewy Bodies (DLB). Recently, microRNAs (miRNAs) have received a lot of attention as the novel biomarkers for dementia. Here, using serum miRNA expression of 1,601 Japanese individuals, we investigated potential miRNA biomarkers and constructed risk prediction models, based on a supervised principal component analysis (PCA) logistic regression method, according to the subtype of dementia. The final risk prediction model achieved a high accuracy of 0.873 on a validation cohort in AD, when using 78 miRNAs: Accuracy = 0.836 with 86 miRNAs in VaD; Accuracy = 0.825 with 110 miRNAs in DLB. To our knowledge, this is the first report applying miRNA-based risk prediction models to a dementia prospective cohort. Our study demonstrates our models to be effective in prospective disease risk prediction, and with further improvement may contribute to practical clinical use in dementia

Use of Non-Amplified RNA Samples for Microarray Analysis of Gene Expression

Demand for high quality gene expression data has driven the development of revolutionary microarray technologies. The quality of the data is affected by the performance of the microarray platform as well as how the nucleic acid targets are prepared. The most common method for target nucleic acid preparation includes in vitro transcription amplification of the sample RNA. Although this method requires a small amount of starting material and is reported to have high reproducibility, there are also technical disadvantages such as amplification bias and the long, laborious protocol. Using RNA derived from human brain, breast and colon, we demonstrate that a non-amplification method, which was previously shown to be inferior, could be transformed to a highly quantitative method with a dynamic range of five orders of magnitude. Furthermore, the correlation coefficient calculated by comparing microarray assays using non-amplified samples with qRT-PCR assays was approximately 0.9, a value much higher than when samples were prepared using amplification methods. Our results were also compared with data from various microarray platforms studied in the MicroArray Quality Control (MAQC) project. In combination with micro-columnar 3D-Gene™ microarray, this non-amplification method is applicable to a variety of genetic analyses, including biomarker screening and diagnostic tests for cancer

Genetic polymorphism of pleiotrophin is associated with pain experience in Japanese adults Case-control study

Genetic factors play a role in individual differences in pain experience. Here, we performed a genome-wide association study (GWAS) to identify novel loci regulating pain processing. We conducted a 2-stage GWAS and the candidate single-nucleotide polymorphisms (SNPs) association study on pain experience using an exploratory cohort of patients with cancer pain. The confirmatory cohort comprised of participants from the general population with and without habitual use of analgesic medication. In the exploratory cohort, we evaluated pain intensity using a numerical rating scale, recorded daily opioid dosages, and calculated pain reduction rate. In the confirmatory cohort, pain experience was defined as habitual nonsteroidal anti-inflammatory drug usage. Using linear regression models, we identified candidate SNP in the exploratory samples, and tested the association between phenotype and experienced pain in the confirmatory samples. We found 1 novel SNP (rs11764598)—located on the gene encoding for pleiotrophin on chromosome 7—that passed the genome-wide suggestive significance at 20% false discovery rate (FDR) correction in the exploratory samples of patients with cancer pain (P = 1.31 × 10-7, FDR = 0.101). We confirmed its significant association with daily analgesic usage in the confirmatory cohort (P = .028), although the minor allele affected pain experience in an opposite manner. We identified a novel genetic variant associated with pain experience. Further studies are required to validate the role of pleiotrophin in pain processing

High-Throughput MicroRNA (miRNAs) Arrays Unravel the Prognostic Role of MiR-211 in Pancreatic Cancer

BACKGROUND: Only a subset of radically resected pancreatic ductal adenocarcinoma (PDAC) patients benefit from chemotherapy, and identification of prognostic factors is warranted. Recently miRNAs emerged as diagnostic biomarkers and innovative therapeutic targets, while high-throughput arrays are opening new opportunities to evaluate whether they can predict clinical outcome. The present study evaluated whether comprehensive miRNA expression profiling correlated with overall survival (OS) in resected PDAC patients. METHODOLOGY/PRINCIPAL FINDINGS: High-resolution miRNA profiles were obtained with the Toray's 3D-Gene™-miRNA-chip, detecting more than 1200 human miRNAs. RNA was successfully isolated from paraffin-embedded primary tumors of 19 out of 26 stage-pT3N1 homogeneously treated patients (adjuvant gemcitabine 1000 mg/m(2)/day, days-1/8/15, every 28 days), carefully selected according to their outcome (OS<12 (N = 13) vs. OS>30 months (N = 6), i.e. short/long-OS). Highly stringent statistics included t-test, distance matrix with Spearman-ranked correlation, and iterative approaches. Unsupervised hierarchical analysis revealed that PDACs clustered according to their short/long-OS classification, while the feature selection algorithm RELIEF identified the top 4 discriminating miRNAs between the two groups. These miRNAs target more than 1500 transcripts, including 169 targeted by two or more. MiR-211 emerged as the best discriminating miRNA, with significantly higher expression in long- vs. short-OS patients. The expression of this miRNA was subsequently assessed by quantitative-PCR in an independent cohort of laser-microdissected PDACs from 60 resected patients treated with the same gemcitabine regimen. Patients with low miR-211 expression according to median value had a significantly shorter median OS (14.8, 95%CI = 13.1-16.5, vs. 25.7 months, 95%CI = 16.2-35.1, log-rank-P = 0.004). Multivariate analysis demonstrated that low miR-211 expression was an independent factor of poor prognosis (hazard ratio 2.3, P = 0.03) after adjusting for all the factors influencing outcome. CONCLUSIONS/SIGNIFICANCE: Through comprehensive microarray analysis and PCR validation we identified miR-211 as a prognostic factor in resected PDAC. These results prompt further prospective studies and research on the biological role of miR-211 in PDAC

Carbon Ion Irradiation Activates Anti-Cancer Immunity

Carbon ion beams have the unique property of higher linear energy transfer, which causes clustered damage of DNA, impacting the cell repair system. This sometimes triggers apoptosis and the release in the cytoplasm of damaged DNA, leading to type I interferon (IFN) secretion via the activation of the cyclic GMP–AMP synthase-stimulator of interferon genes pathway. Dendritic cells phagocytize dead cancer cells and damaged DNA derived from injured cancer cells, which together activate dendritic cells to present cancer-derived antigens to antigen-specific T cells in the lymph nodes. Thus, carbon ion radiation therapy (CIRT) activates anti-cancer immunity. However, cancer is protected by the tumor microenvironment (TME), which consists of pro-cancerous immune cells, such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The TME is too robust to be destroyed by the CIRT-mediated anti-cancer immunity. Various modalities targeting regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages have been developed. Preclinical studies have shown that CIRT-mediated anti-cancer immunity exerts its effects in the presence of these modalities. In this review article, we provide an overview of CIRT-mediated anti-cancer immunity, with a particular focus on recently identified means of targeting the TME