Increased expression of cell-cell signaling genes by stimulated mononuclear leukocytes in patients with previous atherothrombotic stroke. A whole genome expression profile study.

Background/aims: Inflammation plays an important role in atherosclerosis and stroke. Acute infections are recognized as trigger factors for ischemic stroke.

Methods: In this whole genome expression profile study of 15 patients and 15 control subjects, we tested the hypothesis that patients with a history of atherothrombotic stroke show enhanced transcription of inflammatory genes in circulating leukocytes.

Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels.

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body.

Human p53 knock-in (hupki) mice do not differ in liver tumor response from their counterparts with murine p53.

Mouse models are important tools in toxicologic research. Differences between species in pathways contributing to tumor development, however, raise the question in how far mouse models are valid for human risk assessment. One striking difference relates to the frequency of spontaneous liver cancer which is high in certain mouse strains but rather low in humans.

p53 mutations in benzo(a)pyrene-exposed human p53 knock-in murine fibroblasts correlate with p53 mutations in human lung tumors.

Human p53 mutation spectra differ significantly from one cancer type to another. One possible reason is that carcinogenic risk factors differ, and these factors elicit distinct mutation patterns. There has been no mammalian assay, however, with which to generate mutation patterns in human p53 sequences experimentally, hampering interpretation of the human tumor spectra.

Expression profiling of human hepatoma cells reveals global repression of genes involved in cell proliferation, growth, and apoptosis upon infection with parvovirus H-1.

Autonomous parvoviruses are characterized by their stringent dependency on host cell S phase and their cytopathic effects on neoplastic cells. To better understand the interactions between the virus and its host cell, we used oligonucleotide arrays that carry more than 19,000 unique human gene sequences to profile the gene expression of the human hepatocellular carcinoma cell line QGY-7703 at two time points after parvovirus H-1 infection. At the 6-h time point, a single gene was differentially expressed with a >2.

Head and neck tumor sites differ in prevalence and spectrum of p53 alterations but these have limited prognostic value.

The tumor site is a strong clinical factor in head and neck squamous cell carcinoma (HNSCC). To clarify the biologic and clinical role of p53 alterations in HNSCC, we have examined the prevalence and the nature of p53 alterations in a large cohort of tumors from the different sites. For immunohistochemical analysis of p53 protein expression, we introduced tyramide signal amplification immunohistochemistry (TSA-IHC) on a tissue microarray.

p53 designer genes for the modern mouse.

Major efforts are underway to develop molecular strategies that target the p53 pathway for the treatment of cancer. Mouse strains with humanized p53 sequences that present the precise human DNA-binding domain as mutation target could be informative models to test p53 rescue drugs, and to explore experimentally the causes of human tumor mutations.

High-accuracy amplification of nanogram total RNA amounts for gene profiling.

Microarray-based gene profiling of laser-assisted microdissected tissues or clinical biopsies is still a challenge since the amount of total RNA in such samples is limited and amplification of RNA is mandatory. Representative amplification of mRNA is highly dependent on the reverse transcription reaction, which is error prone, and on the number of amplification cycles. To improve the accuracy of RNA amplification, we optimized, combined, and tested different amplification strategies for Affymetrix oligonucleotide array hybridization.

Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene.

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m(2)) were passaged >20 times.

Medullary epithelial cells of the human thymus express a highly diverse selection of tissue-specific genes colocalized in chromosomal clusters.

Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes.

Analysis of CREM-dependent gene expression during mouse spermatogenesis.

The transcription factors CREM, CREB, and ATF-1 constitute a subfamily of beta-Zip transcription factors. Several different kinase cascades regulate the activity of these proteins. The activator splice-isoform CREMtau is specifically and highly expressed in post-meiotic germ cells during mouse spermatogenesis.

Laser-controlled microdissection of tissues opens a window of new opportunities.

Gene expression analysis using total RNA of bulk tissue usually cannot assign specific messages to particular cell types. Cell-specific RNA expression profiling, though, may be crucial for a better understanding ofthe role of each distinct cell type within a physiological or pathophysiological setting. RNA profiling based on laser-controlled microdissection (LCM) of defined cells of a tissue now provides a useful tool for studying molecular crosstalk between different cell types within a tissue.

Cell type- and promoter-specific roles of Ser18 phosphorylation in regulating p53 responses.

Phosphorylation of mouse p53 at Ser18 occurs after DNA damage. To determine the physiological roles of this phosphorylation event in p53-dependent DNA damage responses, a Ser18 to Ala missense mutation was introduced into the germline of mice. Thymocytes and fibroblasts from the knock-in mice show reduced transactivation of many p53 target genes following DNA damage.

Expression profiling of CC531 colon carcinoma cells reveals similar regulation of beta-catenin target genes by both butyrate and aspirin.

The CC531 cell line has been widely used to study different aspects of tumor growth and metastasis and provides an excellent experimental platform to develop novel antitumor strategies. To characterize the CC531 model at the molecular level, we screened for mutations in genes covering important signal-transduction pathways that are known to play major roles during colon carcinogenesis, the wnt and the ki-ras signaling pathways. We found both a prototypic beta-catenin (Ctnnb1) mutation (Thr(41)Ile) and a ki-ras (G12D) mutation, providing unambiguous evidence for the constitutive activation of these pathways in CC531 cells.

Induction of Epstein-Barr virus (EBV) reactivation in Raji cells by doxorubicin and cisplatin.

Background: Epstein-Barr virus (EBV) can be activated in B-lymphoid cells to enter the lytic cycle by various kinds of stimuli, including 12-O-tetradecanoylphorbol-1 3-acetate (TPA), butyric acid, calcium ionophore A23187, transforming growth factor-beta and anti-immunoglobulin crosslinking. EBV reactivation has been clinically observed in patients receiving systemic chemotherapy. This study sought in vitro evidence to suggest whether anticancer drugs may directly contribute to the EBV reactivation.

Mouse models for generating P53 gene mutation spectra.

The p53 tumor suppressor gene lends itself to mutation spectra analysis, because the frequency of point mutations in human tumors is high, the locations of inactivating tumor mutations are numerous and dispersed, and all possible base substitutions are observed in human cancer. P53 tumor mutations induced experimentally in mice exposed to carcinogens have been described, but have not yet contributed significantly to our understanding of mutagenic mechanisms or of the origins of mutations in human cancers. Recently, gene-targeting technology has allowed development of a new mouse model, which explores experimentally the endogenous and environmental factors that may contribute to neoplastic disease in humans.

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed.

The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells.

To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency.