The remarkable generation of scores of increasingly sophisticated mouse models of mammary cancer over the past two decades has provided tremendous insights into molecular derangements that can lead to cancer.The relationships of these models to human breast cancer, however, remain problematic.Recent advances in genomic technologies offer significant opportunities to identify critical changes that occur during cancer evolution and to distinguish in a complex and comprehensive manner the key similarities and differences between mouse models and human cancer.Comparisons between mouse and human tumors are being performed using comparative genomic hybridization, gene expression profiling, and proteomic analyses.The appropriate use of genetically engineered mouse models of mammary cancer in preclinical studies remains an important challenge which may also be aided by genomic technologies.Genomic approaches to cancer are generating huge datasets that represent a complex system of underlying networks of genetic interactions.Mouse models offer a tremendous opportunity to identify such networks and how they relate to human cancer.The challenge of the future remains to decipher these networks in order to identify the genetic nodes of oncogenesis that may be important targets for chemoprevention and therapy.

The objective of this study was to use gene expression data from well-defined cell culture models, in combination with expression data from diagnostic samples of human diseased tissues, to identify potential therapeutic targets and markers of disease. Using Affymetrix oligonucleotide array technology, we identified a common profile of genes upregulated during endothelial morphogenesis into tubelike structures in three in vitro models of angiogenesis. Rigorous data selection criteria were used to identify a list of over 1,000 genes whose expression was increased more than twofold over baseline at either 4, 8, 24, 40 or 50 h. To further refine and prioritize this list, we used standard bioinformatic algorithms to identify potential transmembrane and secreted proteins. We then overlapped this gene set with genes upregulated in colon tumors vs. normal colon, resulting in a subset of 128 genes in common with our endothelial list. We removed from this list those genes expressed in 6 different colon tumor lines, resulting in a list of 24 putative, vascular-specific angiogenesis-associated genes. Three genes, gp34, stanniocalcin-1 (STC-1), and GA733-1, were expressed at levels 10-fold or more in colon tumors compared with normal mucosa. We validated the vascular-specific expression of one of these genes, STC-1, by in situ hybridization. The ability to combine in vitro and in vivo data sets should permit one to identify putative angiogenesis target genes in various tumors, chronic inflammation, and other disorders where therapeutic manipulation of angiogenesis is a desirable treatment modality.

Novel drug targets can be identified by differential analysis of RNA transcripts isolated from cancer cell lines and tissues. We have extended this approach by analyzing differences in gene expression resulting from the drug treatment of transformed and nontransformed cells. A mouse mammary epithelial cell line (C57MG), which conditionally expresses the Wnt-1 proto-oncogene, was left untreated or treated with retinoic acid in the presence or absence of Wnt-1 expression. The experiment was performed in triplicate, and RNA extracted from the four samples was analyzed by hybridization to over 12,000 unique oligonucleotide probe sets. Reproducible alterations in gene expression that occurred in response to retinoic acid, Wnt-1, or retinoic acid plus Wnt-1 relative to untreated cells were identified. Greater attention was given to genes encoding cell surface antigens that were selectively up-regulated by the combination of Wnt-1 and retinoic acid. These genes included the tumor necrosis factor family 4-1BB ligand, ephrin B1, stra6, autotaxin, and ISLR. Administration of retinoic acid to mice bearing tumors driven by activation of the Wnt-1/beta-catenin pathway resulted in increased expression of stra6 in the tumors but not in normal tissue. In principal, the therapeutic index of antibodies directed against these antigens should be enhanced by co-administration of retinoic acid.

This chapter describes application of immunoadhesins, perhaps most relevant for the behavioral neuroscientist. It focuses on a particular protein family (EphA receptor tyrosine kinases) and the investigation of their potential role in certain aspects of behavior and brain function. The chapter is intended to be an example to delineate the advantages along with the disadvantages of using immunoadhesins, a method novel in behavioral neuroscience. In addition, the present findings revealed a role for EphA receptor tyrosine kinases in cognitive function in the adult mammalian brain. These findings open an unexpected avenue into the functional analysis of this large receptor protein family and may lead to novel targets for therapeutic intervention in human brain and behavioral disorders. They also demonstrate that protein targeting with the application of immunoadhesins may be a useful research strategy in the analysis of molecular components involved in such complex behavioral traits as learning and memory.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

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.

EphA family receptor tyrosine kinases and their ephrin-A ligands are involved in patterning axonal connections during brain development, but until now a role for these molecules in the mature brain had not been elucidated. Here, we show that both the EphA5 receptor and its ephrin-A ligands (2 and 5) are expressed in the adult mouse hippocampus, and the EphA5 protein is present in a phosphorylated form. Because there are no pharmacological agents available for EphA receptors, we designed recombinant immunoadhesins that specifically bind to the receptor binding site of the ephrin-A ligand (antagonist) or the ligand binding site of the EphA receptor (agonist) and thus target EphA function. We demonstrate that intrahippocampal infusion of an EphA antagonist immunoadhesin leads to impaired performance in two behavioral paradigms, T-maze spontaneous alternation and context-dependent fear conditioning, sensitive to hippocampal function, whereas activation of EphA by infusion of an agonist immunoadhesin results in enhanced performance on these tasks. Because the two behavioral tasks have different motivational, perceptual, and motor requirements, we infer the changes were not caused by these performance factors but rather to cognitive alterations. We also find bidirectional changes in gene expression and in electrophysiological measures of synaptic efficacy that correlate with the behavioral results. Thus, EphA receptors and their ligands are implicated as mediators of plasticity in the adult mammalian brain.

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.

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.

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.

Endothelin, a potent vasoconstrictor, is produced by cardiac fibroblasts in culture and induces hypertrophy in cardiac myoctes. The purpose of this study was to determine whether vasoactive factors endogenous to the heart affect the production of endothelin by cultured cardiac fibroblasts. Vasoactive factors have been shown to play multiple roles in the adaptation of the heart to chronic overload, affecting both vascular tone and cell growth. Both atrial (ANP) and brain (BNP) natriuretic peptides are endogenous cardiac vasodilators and are produced by cultured myocytes in response to stimulation with endothelin. Treatment of cardiac fibroblasts with these peptides decreased endothelin production. Nitroprusside, an activator of guanylyl cyclase, decreased endothelin production indicating the involvement of cGMP in the response. Carbaprostacyclin, a stable derivative of prostacyclin, another endogenous cardiac vasodilator, also decreased endothelin production by fibroblasts. The combination of BNP and carbaprostacyclin was additive in decreasing endothelin production. In contrast, PGF2α and angiotensin II, both endogenous cardiac vasoconstrictors, increased endothelin production and overcame the inhibition induced by BNP and carba-prostacyclin. In summary, endothelin production by cardiac fibroblasts was decreased by the endogenous cardiac vasodilators ANP, BNP, and prostacyclin and increased by the endogenous vasoconstrictors PGF2α and angiotensin II.