Formoterol fumarate and roxithromycin effects on muscle mass in an animal model of cancer cachexia.

Our study probed the effects of the beta-2 adrenergic agonist, formoterol and the macrolide antibiotic, roxithromycin, on muscle wasting in a well-characterized animal model of cancer cachexia. Female Wistar rats were inoculated with Yoshida AH130 ascites hepatoma (AH) cells to induce rapid and severe cachexia as demonstrated by wet weight determinations of the hearts, gastrocnemius muscles and carcasses. The control animals received saline (vehicle) inoculations.

A single nucleotide polymorphism based approach for the identification and characterization of gene expression modulation using MassARRAY.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. Their abundance and the ease with which they can be assayed have lead to their use in applications beyond simple genotyping. One such application is the quantitative determination of transcript levels associated with distinct alleles or haplotypes found in promoters and coding regions of genes.

Role of ICAM1 in invasion of human breast cancer cells.

We identified previously a region on chromosome 19p13.2 spanning the genes encoding the intercellular adhesion molecules (ICAM), ICAM1, ICAM4 and ICAM5 as a breast cancer susceptibility locus. Genetic variants in this region were also associated with indicators of disease severity, including higher rates of metastases to other organs.

Suppression of RAD21 gene expression decreases cell growth and enhances cytotoxicity of etoposide and bleomycin in human breast cancer cells.

A genome-wide case-control association study done in our laboratory has identified a single nucleotide polymorphism located in RAD21 as being significantly associated with breast cancer susceptibility. RAD21 is believed to function in sister chromatid alignment as part of the cohesin complex and also in double-strand break (DSB) repair. Following our initial finding, expression studies revealed a 1.

Mining disease susceptibility genes through SNP analyses and expression profiling using MALDI-TOF mass spectrometry.

To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis.

Expression of base excision DNA repair genes is a sensitive biomarker for in vivo detection of chemical-induced chronic oxidative stress: identification of the molecular source of radicals responsible for DNA damage by peroxisome proliferators.

Oxidative stress to DNA is recognized as one of the mechanisms for the carcinogenic effects of some environmental agents. Numerous studies have been conducted in an attempt to document the fact that chemical carcinogens that are thought to induce production of oxidants also cause the formation of oxidative DNA lesions. Although many DNA adducts continue to be useful biomarkers of dose/effect, changes in gene expression have been proposed to be a practical novel tool for studying the role of chemically induced oxidative DNA damage.

Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF.

The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation.

Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers.

It is estimated that cigarette smoking kills over 1 000 000 people each year by causing lung cancer as well as many other neoplasmas. p53 mutations are frequent in tobacco-related cancers and the mutation load is often higher in cancers from smokers than from nonsmokers. In lung cancers, the p53 mutational patterns are different between smokers and nonsmokers with an excess of G to T transversions in smoking-associated cancers.

Mapping polycyclic aromatic hydrocarbon and aromatic amine-induced DNA damage in cancer-related genes at the sequence level.

Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis. In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair. Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential.

Matrix-induced fragmentation of P3'-N5' phosphoramidate-containing DNA: high-throughput MALDI-TOF analysis of genomic sequence polymorphisms.

Chemical and enzymatic approaches were used to produce polynucleotide fragments containing acid-labile internucleotide P3'-N5' phosphoramidate bonds, either in a surface-bound form or in solution. The primer extension reaction utilizing 5'-amino-5'-deoxynucleoside 5'-triphosphates generates polynucleotides that can be fragmented into short, easy-to-analyze pieces simply by being premixed with the acidic matrices typically used for MALDI-TOF mass spectrometry of nucleic acids. This leads to detection procedures that are simple, robust and easy to automate.

Expression of base excision repair enzymes in rat and mouse liver is induced by peroxisome proliferators and is dependent upon carcinogenic potency.

Elevated and sustained cell replication, together with a decrease in apoptosis, is considered to be the main mechanism of hepatic tumor promotion due to peroxisome proliferators. In contrast, the role of oxidative stress and DNA damage in the carcinogenic mechanism is less well understood. In view of possible induction of DNA damage by peroxisome proliferators, DNA repair mechanisms may be an important factor to consider in the mechanism of action of these compounds.

Promoter structure and cell cycle dependent expression of the human methylpurine-DNA glycosylase gene.

The methylpurine-DNA glycosylase (MPG) gene coding for human 3-methyladenine (3-meAde)-DNA glycosylase functions in the first step of base excision repair (BER) to remove numerous damaged bases including 3-meGua, ethenoadenine, and hypoxanthine (Hx) in addition to 3-meAde. In this report, we identify the length of the minimal MPG promoter region, demonstrate the involvement of several transcription factors in expression of the MPG gene, and determine the point at which transcription initiates. We also demonstrate that control of MPG expression is linked to MPG activity.

Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons.

Background: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in combustion products of organic matter, including cigarette smoke. Metabolically activated diol epoxides of these compounds, including benzo[a]pyrene diol epoxide (B[a]PDE), have been suggested as causative agents in the development of lung cancer. We previously mapped the distribution of B[a]PDE adducts within the p53 tumor suppressor gene (also known as TP53), which is mutated in 60% of human lung cancers, and found that B[a]PDE adducts preferentially form at lung cancer mutational hotspots (codons 154, 157, 158, 245, 248, and 273).

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers.

Quantitation and mapping of aflatoxin B1-induced DNA damage in genomic DNA using aflatoxin B1-8,9-epoxide and microsomal activation systems.

Aflatoxin B1 (AFB1) is a mutagenic and carcinogenic mycotoxin which may play a role in the etiology of human liver cancer. In vitro studies have shown that AFB1 adducts form primarily at the N7 position of guanine. Using quantitative PCR (QPCR) and ligation-mediated PCR (LMPCR), we have mapped total AFB1 adducts in genomic DNA treated with AFB1-8,9-epoxide and in hepatocytes exposed to AFB1 activated by rat liver microsomes or human liver and enterocyte microsomal preparations.

PCR-based approaches to adduct analysis.

Ligation-mediated polymerase chain reaction (LMPCR) is a PCR-based method for the detection of DNA adducts at individual nucleotide positions in mammalian genes. Adduct-specific enzymes, such as T4 endonuclease V, various base excision repair enzymes, UvrABC nuclease, and chemical cleavage techniques can be used to convert the adducts into DNA strand breaks. The positions of these breaks are then detected by LMPCR.

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts.

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors.

Formation and repair of DNA lesions in the p53 gene: relation to cancer mutations?

The number and diversity of mutations in the p53 mutation data base provides indirect evidence that implicates environmental mutagens in human carcinogenesis. The p53 gene has a large mutational target size; more than 280 out of 393 amino acids are found mutated in tumors. We argue that there is possibly a limited involvement of selection for specific mutations in the central domain of the protein, and that the distribution of DNA damage along the p53 gene caused by environmental carcinogens can be correlated with the mutational spectra, i.

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts.

Sunlight induces pyrimidine dimers preferentially at 5-methylcytosine bases.

The most prevalent DNA lesion induced by UV irradiation is the cyclobutane pyrimidine dimer (CPD), which forms at positions of neighboring pyrimidines. Here we show that the rare DNA base 5-methylcytosine is the preferred target for CPD formation when cells are irradiated with natural sunlight. We have mapped the distribution of CPDs formed in normal human keratinocytes along exons of the p53 gene.

Cytosine methylation determines hot spots of DNA damage in the human P53 gene.

In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F.

Modulation of (+/-)-anti-BPDE mediated p53 accumulation by inhibitors of protein kinase C and poly(ADP-ribose) polymerase.

The rapid accumulation of the p53 gene product is considered to be an important component of the cellular response to a variety of genotoxins. In order to gain insights on the biochemical pathways leading to p53 stabilization, the effect of (+/-) 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)-pyrene [(+/-)-anti-BPDE] induced DNA damage on p53 protein levels was investigated in various repair-proficient and repair-deficient human cells. Brief exposure of normal human fibroblasts to 0.

Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53.

Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273.