Effects of hyaluronic acid and CD44 interaction on the proliferation and invasiveness of malignant pleural mesothelioma

Hyaluronic acid (HA) has been proposed as a biochemical marker of malignant pleural mesothelioma (MPM). The present study focused on the implications of HA and CD44 interaction in the proliferation and invasiveness of MPM. The proliferation and invasive activity was evaluated in two human mesothelioma cell lines, ACC-MESO-1 and K921MSO, by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the transwell chamber model. The knockdown of CD44 gene expression was accomplished by transfection of the cells with small interfering RNA. Flow cytometry revealed that both the ACC-MESO-1 and K921MSO cell lines highly expressed CD44. Treatment with HA enhanced the proliferation in both mesothelioma cell lines in comparison to cells without HA treatment. The treatment with HA (25???g/ml) also significantly upregulated the invasion of both types of cells. The silencing of CD44 significantly abrogated the effect of HA treatment on the proliferation of ACC-MESO-1 cells and significantly suppressed the proliferation of K921MSO cells. HA?CCD44 binding is important for the migration and proliferation of mesothelioma cells. Therefore, the HA?CCD44 interaction is a potentially useful therapeutic target in MPM.     

High-throughput resequencing of target-captured cDNA in cancer cells

The recent advent of whole exon (exome)-capture technology, coupled with second-generation sequencers, has made it possible to readily detect genomic alterations that affect encoded proteins in cancer cells. Such target resequencing of the cancer genome, however, fails to detect most clinically-relevant gene fusions, given that such oncogenic fusion genes are often generated through intron-to-intron ligation. To develop a resequencing platform that simultaneously captures point mutations, insertions-deletions (indels), and gene fusions in the cancer genome, we chose cDNA as the input for target capture and extensive resequencing, and we describe the versatility of such a cDNA-capture system. As a test case, we constructed a custom target-capture system for 913 cancer-related genes, and we purified cDNA fragments for the target gene set from five cell lines of CML. Our target gene set included Abelson murine leukemia viral oncogene homolog 1 (ABL1), but it did not include breakpoint cluster region (BCR); however, the sequence output faithfully detected reads spanning the fusion points of these two genes in all cell lines, confirming the ability of cDNA capture to detect gene fusions. Furthermore, computational analysis of the sequence dataset successfully identified non-synonymous mutations and indels, including those of tumor protein p53 (TP53). Our data might thus support the feasibility of a cDNA-capture system coupled with massively parallel sequencing as a simple platform for the detection of a variety of anomalies in protein-coding genes among hundreds of cancer specimens.     

Combination of ADH1B*2/ALDH2*2 polymorphisms alters acetaldehyde-derived DNA damage in the blood of Japanese alcoholics

The acetaldehyde associated with alcoholic beverages is an evident carcinogen for the esophagus. Genetic polymorphisms of the alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) genes are associated with the risk of esophageal cancer. However, the exact mechanism via which these genetic polymorphisms affect esophageal carcinogenesis has not been elucidated. ADH1B*2 is involved in overproduction of acetaldehyde due to increased ethanol metabolism into acetaldehyde, and ALDH2*2 is involved in accumulation of acetaldehyde due to the deficiency of acetaldehyde metabolism. Acetaldehyde can interact with DNA and form DNA adducts, resulting in DNA damage. N²‐ethylidene‐2′‐deoxyguanosine (N²‐ethylidene‐dG) is the most abundant DNA adduct derived from acetaldehyde. Therefore, we quantified N²‐ethylidene‐dG levels in blood samples from 66 Japanese alcoholic patients using liquid chromatography/electrospray tandem mass spectrometry, and investigated the relationship between N²‐ethylidene‐dG levels and ADH1B and ALDH2 genotypes. The median N²‐ethylidene‐dG levels (25th percentile, 75th percentile) in patients with ADH1B*1/*1 plus ALDH2*1/*1, ADH1B*2 carrier plus ALDH2*1/*1, ADH1B*1/*1 plus ALDH2*1/*2, and ADH1B*2 carrier plus ALDH2*1/*2 were 2.14 (0.97, 2.37)/10⁷ bases, 2.38 (1.18, 2.98)/10⁷ bases, 5.38 (3.19, 6.52)/10⁷ bases, and 21.04 (12.75, 34.80)/10⁷ bases, respectively. In the ALDH2*1/*2 group, N²‐ethylidene‐dG levels were significantly higher in ADH1B*2 carriers than in the ADH1B*1/*1 group (P < 0.01). N²‐ethylidene‐dG levels were significantly higher in the ALDH2*1/*2 group than in the ALDH2*1/*1 group, regardless of ADH1B genotype (ADH1B*1/*1, P < 0.05; ADH1B*2 carriers, P < 0.01) N²‐ethylidene‐dG levels in blood DNA of the alcoholics was remarkably higher in individuals with a combination of the ADH1B*2 and ALDH2*2 alleles. These results provide a new perspective on the carcinogenicity of the acetaldehyde associated with alcoholic beverages, from the aspect of DNA damage.     

Antitumor activity of human γδ T cells transducted with CD8 and with T-cell receptors of tumor-specific cytotoxic T lymphocytes

The difficulty in the induction and preparation of a large number of autologous tumor-specific cytotoxic T lymphocytes (CTL) from individual patients is one of major problems in their application to adoptive immunotherapy. The present study tried to establish the useful antitumor effectors by using γδ T cells through tumor-specific TCRαβ genes transduction, and evaluated the efficacy of their adoptive transfer in a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice model. The TCRαβ gene was cloned from the HLA-B15-restricted CTL clone specific of the Kita-Kyushu Lung Cancer antigen-1 (KK-LC-1). The cloned TCRαβ as well as the CD8 gene were transduced into γδ T cells induced from peripheral blood lymphocytes (PBL). Cytotoxic T lymphocyte activity was examined using a standard 4 h (51) Cr release assay. Mice with a xenotransplanted tumor were treated with an injection of effector cells. Successful transduction of TCRαβ was confirmed by the staining of KK-LC-1-specific tetramers. The γδ T cells transduced with TCRαβ and CD8 showed CTL activity against the KK-LC-1-positive lung cancer cell line in a HLA B15-restricted manner. Adoptive transfer of the effector cells in a mice model resulted in marked growth suppression of KK-LC-1- and HLA-B15-positive xenotransplanted tumors. Co-transducing TCRαβ and CD8 into γδ T cells yielded the same antigen-specific activity as an original CTL in vitro and in vivo. The TCRαβ gene transduction into γδ T cells is a promising strategy for developing new adoptive immunotherapy.     

Coexpression of MUC16 and mesothelin is related to the invasion process in pancreatic ductal adenocarcinoma

The invasion process is a crucial step for pancreatic ductal adenocarcinoma (PDAC); however, the genes related to invasion remain unclear. To identify specific genes for the invasion process, we compared microarray data for infiltrating cancer and PanIN-3, which were harvested from an individual PDAC patient by microdissection. Furthermore, immunohistochemical, coimmunoprecipitation and invasion analyses were performed to confirm the biologic significance of molecules identified by expression profile. In the present study, we focused on MUC16 and mesothelin among 87 genes that were significantly upregulated in infiltrating components compared to PanIN-3 in all PDAC patients, because MUC16 was the most differently expressed between two regions, and mesothelin was reported as the receptor for MUC16. Immunohistochemical analysis revealed that MUC16 and mesothelin were expressed simultaneously only in infiltrating components and increased at the invasion front, and binding of MUC16 and mesothelin was found in PDAC by immunoprecipitation assay. The downregulation of MUC16 by shRNA and the blockage of MUC16 binding to mesothelin by antibody inhibited both invasion and migration of pancreatic cancer cell line. MUC16 high/mesothelin high expression was an independent prognostic factor for poor survival in PDAC patients. In conclusion, we identified two specific genes, MUC16 and mesothelin, associated with the invasion process in patients with PDAC.     

Assessing treatment effects of inhaled corticosteroids on medical expenses and exacerbations among COPD patients: longitudinal analysis of managed care claims

Objective. To assess costs, effectiveness, and cost‐effectiveness of inhaled corticosteroids (ICS) augmenting bronchodilator treatment for chronic obstructive pulmonary disease (COPD). Data Sources. Claims between 1997 and 2005 from a large managed care database. Study Design. Individual‐level, fixed‐effects regression models estimated the effects of initiating ICS on medical expenses and likelihood of severe exacerbation. Bootstrapping provided estimates of the incremental cost per severe exacerbation avoided. Data Extraction Methods. COPD patients aged 40 or older with ≥15 months of continuous eligibility were identified. Monthly observations for 1 year before and up to 2 years following initiation of bronchodilators were constructed. Principal Findings. ICS treatment reduced monthly risk of severe exacerbation by 25 percent. Total costs with ICS increased for 16 months, but declined thereafter. ICS use was cost saving 46 percent of the time, with an incremental cost‐effectiveness ratio of $2,973 per exacerbation avoided; for patients ≥50 years old, ICS was cost saving 57 percent of time. Conclusions. ICS treatment reduces exacerbations, with an increase in total costs initially for the full sample. Compared with younger patients with COPD, patients aged 50 or older have reduced costs and improved outcomes. The estimated cost per severe exacerbation avoided, however, may be high for either group because of uncertainty as reflected by the large standard errors of the parameter estimates.     

Nicotine inhibits mineralization of human dental pulp cells

Nicotine is a major component of tobacco smoke, and signals via nicotinic acetylcholine receptors (nAChR). However, little is known about the effects of nicotine on human dental pulp cells (HDPCs). In this study, we assessed the effects of nicotine on mineralization in HDPCs. We confirmed messenger RNA expression of nAChR subunits and examined the effects of nicotine on expression of extracellular matrices (ECMs), alkaline phosphatase (ALP) activity, and mineralized nodule formation by HDPCs. Gene expression of nAChR subunits alpha1, alpha2, alpha 4, alpha 5, alpha 6, alpha 7, beta1, beta2, and beta 4 was detected in HDPCs. Interestingly, the messenger RNA expression of dentin matrix acidic phosphoprotein-1, bone sialoprotein, and ALP activity were significantly reduced in nicotine-treated HDPC. In addition, mineralized nodule formation, which was examined by alizarin red staining, was also inhibited in HDPCs by the same treatment. These results indicate that nicotine suppresses the cytodifferentiation and mineralization of HDPCs, possibly via nAChR.