P. Mickey Williams

Expression profiling of a human cell line model of prostatic cancer reveals a direct involvement of interferon signaling in prostate tumor progression

Cancer-associated fibroblasts induce malignant behavior in genetically initiated but nontumorigenic human prostatic epithelium. The genetic basis for such transformation is still unknown. By using Affymetrix GeneChip technology, we profiled genomewide gene expression of transformed [tumorigenic benign prostatic hyperplasia (BPH1)CAFTD] and parental (nontumorigenic BPH1) cells. We identified differentially expressed genes, which are associated with tumorigenesis or tumor progression. One striking finding is that a significant portion of the down-regulated genes belongs to interferon (IFN)-inducible molecules. We show that IFN inhibited the tumorigenic BPH1CAFTD cell proliferation and colony formation in vitro and inhibited tumor growth in xenografts in vivo. Expression of the IFN-inducible molecules correlates with the growth-inhibiting effects of IFN. In addition, these genes are reported to be mapped mainly to two chromosomal regions, 10q23–26 and 17q21, which are frequently deleted in human prostate cancers. Furthermore, in silico data-mining with the GeneLogic database revealed that expression of the IFN-inducible genes was down-regulated in approximately 30% of the 49 clinically characterized samples of prostatic adenocarcinomas. Collectively, we show that there seems to be a direct link between IFN-inducible molecules and prostatic tumor progression. These findings suggest IFN-inducible molecules as potential therapeutic targets for the treatment of prostate cancer.

Vascular Endothelial Growth Factor–Induced Genes in Human Umbilical Vein Endothelial Cells: Relative Roles of KDR and Flt-1 Receptors

Objective—This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. Methods and Results—We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. Conclusions—The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.

Branching Out: A Molecular Fingerprint of Endothelial Differentiation into Tube-Like Structures Generated by Affymetrix Oligonucleotide Arrays

The process of endothelial differentiation into a network of tube‐like structures with patent lumens requires an integrated program of gene expression. To identify genes upregulated in endothelial cells during the process of tube formation, RNA was prepared from several different time points (0, 4, 8, 24, 40, and 48 hours) and from three different experimental models of human endothelial tube formation: in collagen gels and fibrin gels driven by the combination of PMA (80), bFGF (40 ng/ml) and bFGF (40 ng/ml) or in collagen gels driven by the combination of HGF (40 ng/ml) and VEGF (40 ng/ml). Gene expression was evaluated using Affymetrix® Gene Chip® oligonucleotide arrays. Over 1000 common genes were upregulated greater than twofold over baseline at one or more time points in the three different models. In the present study, we discuss the identified genes that could be assigned to major functional classes: apoptosis, cytoskeleton, proteases, matrix, and matrix turnover, pumps and transporters, membrane lipid turnover, and junctional molecules or adhesion proteins.

Phenylephrine, endothelin, prostaglandin F2α, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2α(PGF2α), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2α — and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2α treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2α increased the beat rate to 3.3 times that of control. After exposure to the β-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2α, 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2α by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2α. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal α-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal α-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Cardiac fibroblasts produce leukemia inhibitory factor and endothelin, which combine to induce cardiac myocyte hypertrophy in vitro.

Cardiac fibroblasts in culture produce factor(s) that induce hypertrophy of neonatal rat ventricular myocytes in vitro. As in vivo, the myocyte hypertrophy response in culture is characterized by an increase in cell size and contractile protein content, and by the activation of embryonic genes, including the gene for atrial natriuretic peptide. The purpose of this study was to identify the factor(s) produced by fibroblasts that induce myocyte hypertrophy. The fibroblast hypertrophy activity was inhibited using a combination of the endothelin A receptor blocker BQ-123 and an antibody to leukemia inhibitory factor. The individual antagonists each caused a partial inhibition. The mRNAs for both leukemia inhibitory factor and endothelin were detected by RT-PCR analysis and the concentration of both proteins was determined to be approximately 200 pmol/L in the conditioned medium using immunoassays. Purified leukemia inhibitory factor and endothelin each induced distinctive morphological changes in the myocytes. Their combination generated a different morphology similar to that induced by fibroblast conditioned medium. Each factor also induced atrial natriuretic peptide production, but both were required for the myocytes to produce the levels measured after exposure to fibroblast conditioned medium. These results show that hypertrophy activity produced by cardiac fibroblasts in culture is a result of leukemia inhibitory factor and endothelin.

Development and Application of Real-Time Quantitative PCR

Since the original description of the polymerase chain reaction (PCR), novel applications of this technique have been reported at an exponential rate. The power of the method lies in the ability to analyze minute amounts of samples (e. g., dried blood, microliters of liquid, several cells) or even to detect a single molecule. Virtually every area of biological science has been impacted by PCR, including forensics, genetics, clinical diagnostics, drug discovery, and environmental biology.

Chapter 2.2.4 Application of real-time RT-PCR for quantification of gene expression

Publisher Summary This chapter discusses the application of real-time RT-PCR for quantification of gene expression. The use of real-time RT-PCR methodology permits rapid assay development from validation of RNA sample preparation methods, to validation of the invariance of housekeeping genes. Real-time PCR also provides a quantitative method for moderate to high throughput analysis of gene expression. The ABI instrument permits the analysis of 96 tubes simultaneously, allowing for multiple samples and genes to study in single PCR experiment. The ABI instrument permits the analysis of 96 tubes simultaneously, allowing for multiple samples and genes to be studied in single PCR experiment. This system can produce precise and reliable data which correlates with traditional methods. In addition, the chapter illustrates that real-time RT-PCR technology plays a major role in the future of quantitative gene expression analysis.