José Palacios

Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors.

Because of their similarities, serotonin 5-HT2, 5-HT1C, and the recently described 5-HT2F receptors have been classified as members of the 5-HT2 receptor family, and they have been renamed 5-HT2A, 5-HT2C and 5-HT2B, respectively. The regional distribution and cellular localization of mRNA coding for the members of 5-HT2 receptor family were investigated in consecutive tissue sections from the rat brain by in situ hybridization histochemistry. No evidence for the expression of 5-HT2B receptor was found. High levels of 5-HT2A (formerly 5-HT2) receptor mRNA were observed only in few areas, as the frontal cortex, piriform cortex, ventro-caudal part of CA3, medial mammillary nucleus, the pontine nuclei and the motor cranial nerve nuclei in the brainstem, and the ventral horn of the spinal cord. The distribution of 5-HT2A receptor mRNA is generally in good agreement with that of the corresponding binding sites, although discrepancies were sometimes observed. 5-HT2C (formerly 5-HT1C) mRNA was present at very high levels in the choroid plexuses. However, very high levels were also seen in many other brain regions, as the retrosplenial, piriform and entorhinal cortex, anterior olfactory nucleus, lateral septal nucleus, subthalamic nucleus, amygdala, subiculum and ventral part of CA3, lateral habenula, substantia nigra pars compacta, several brainstem nuclei and the whole grey matter of the spinal cord. These results confirm and extend previous observations that 5-HT2C receptor mRNA is present in many brain areas in addition to those autoradiographically shown to have the corresponding binding sites and that 5-HT2C receptor subtype is a principal 5-HT receptor in the brain. From the comparison between their distributions, 5-HT2A and 5-HT2C receptor mRNAs appeared to be expressed in distinct but overlapping sets of brain regions. Both mRNAs coexisted at high levels in the anterior olfactory nucleus, piriform cortex, endopiriform nucleus, claustrum, pyramidal cell layer of the ventral part of CA3, taenia tecta, substantia nigra pars compacta, and several brainstem nuclei. In other regions both mRNAs were present but with different distributions, as the caudate-putamen. These results are also discussed in relation to the physiological meaning of the existence of two so similar receptor subtypes in the brain.

The binding of serotonergic ligands to the porcine choroid plexus: Characterization of a new type of serotonin recognition site

The kinetic and pharmacological characteristics of the binding of [3H]5-HT (serotonin), [3H]8-OH-DPAT (8-OH-2-di-n-propylaminotetraline), [3H]LSD, [3H]ketanserin and [3H]mesulergine to membranes from frontal cortex, hippocampus and choroid plexus of pig brain were studied. The binding of these ligands to frontal cortex and hippocampus demonstrated the presence of 5-HT1 and 5-HT2 sites in both tissues, although hippocampus was richer in 5-HT1 (subtype 5-HT1A) sites. [3H]5-HT, [3H]mesulergine and [3H]LSD labeled the pig choroid plexus with high affinity. The pharmacological profiles of [3H]5-HT and [3H]mesulergine binding to this tissue were closely comparable. Ligands reported as selective for 5-HT1A, 5-HT1B or 5-HT2 subtypes did not show high affinity for these binding sites. Therefore, these 5-HT binding sites in pig choroid plexus could be named 5-HT1C. Other drugs with a high affinity for these sites were methysergide and mianserine. In pig frontal cortex, [3H]5-HT labeled the different subtypes of 5-HT1 sites. In contrast, [3H]mesulergine bound in pig frontal cortex to a small population of sites with pharmacological properties similar to those of the choroid plexus 5-HT1C sites. Possible physiological functions in which these sites might be involved are discussed.

Quantitative receptor autoradiography using [3H]ultrofilm: application to multiple benzodiazepine receptors.

Radiohistochemical methods utilized to label drug and neurotransmitter receptors in vitro at the light microscopic level are quantitative. In this report, the response of tritium-sensitive sheet film ([3H]Ultrofilm) was characterized under conditions used for the light microscopic localization of neurotransmitter receptors by in vitro autoradiographic techniques. Radioactive standards containing varying concentrations of tritium were prepared from brain tissue and exposed to [3H]Ultrofilm for varying lengths of time, and the response of the film was measured by microdensitometry. A 1n optical density versus 1n radioactivity plot provided a useful linear relationship. Relationships established between radioactivity concentrations, exposure times and optical density were utilized to establish appropriate exposure conditions for several [3H]ligands in brain sections. The accuracy and pharmacological relevance of these methods were tested by studying the regional distribution of multiple benzodiazepine (BZ) receptors, and by analyzing the inhibitory potency of the triazolopyridazine, CL218,872, and methyl-beta-carbolinecarboxylate, two agents which discriminate between type 1 and 2 BZ receptors. The results obtained compared favorably with results previously obtained in tissue homogenates by biochemical methods. Overall, these results have practical implications. For example, quantitative autoradiography can be used to generate accurate kinetic and pharmacological displacement curves for small tissue areas. Also, since the film response curve is not linear with exposure (radioactivity X time), autoradiographs of the same sections can have relatively different density patterns depending on the time of exposure.

Correlation of Snail expression with histological grade and lymph node status in breast carcinomas.

Snail is a zinc finger transcription factor that triggers the epithelial-mesenchymal transition (EMT) by directly repressing E-cadherin expression. Snail is required for mesoderm and neural crest formation during embryonic development and has recently been implicated in the EMT associated with tumour progression. In a series of human breast carcinomas, we have analysed the expression of Snail and that of molecules of the E-cadherin/catenin complexes. We have also correlated these data with the pathological features of the tumours. We show that Snail expression inversely correlates with the grade of differentiation of the tumours and that it is expressed in all the infiltrating ductal carcinomas (IDC) presenting lymph node metastases that were analysed. In addition, Snail is expressed in some dedifferentiated tumours with a negative nodal status. Considering that Snail is involved in the induction of the invasive and migratory phenotype in epithelial cells, these results indicate that it is also involved in the progression of breast ductal tumours, where it could additionally serve as a marker of the metastatic potential.

Localization of 5-HT1B, 5-HT1Dα, 5-HT1E and 5-HT1F receptor messenger RNA in rodent and primate brain

In situ hybridization histochemistry (ISHH) was used to study the distribution of various 5-HT1 receptor messenger RNAs (mRNA) in the mammalian nervous system. Since the cDNAs encoding the different 5-HT1 receptors, have not been cloned in one single species, brains of the species appropriate for the 5-HT1 receptor messenger RNA (mRNA) have been used. Thus, 5-HT1B and 5-HT1D alpha mRNA were determined in rat and mouse brain, while 5-HT1E and 5-HT1F mRNA were studied in human (and monkey) and guinea-pig brain, respectively. 5-HT1B and 5-HT1D alpha hybridization signals were predominantly present in caudate-putamen and cortical areas; in addition, 5-HT1B mRNA was also detected in hippocampus, cerebellum and cerebral arteries. In general, the distribution of 5-HT1B mRNA was characterized by high densities, whereas 5-HT1D alpha mRNA was expressed at very low levels. Comparison of the localization of the mRNAs to the regional distributions of the 5-HT1B and 5-HT1D binding sites in rat brain (described in a previous study), revealed that both receptor subtypes could be putative presynaptic heteroreceptors, modulating the release of various neurotransmitters in the central nervous system. The mRNA encoding the recently cloned 5-HT1E receptor, which has low affinity for the 5-HT1 receptor ligand 5-carboxamidotryptamine (5-CT), was localized in human brain. It was found to be present in cortical areas, caudate, putamen and amygdala, areas known to contain 5-CT insensitive 5-HT1 binding sites. The regional distribution of the 5-HT1F mRNA was determined in guinea-pig brain: high densities were observed in various cortical areas, the hippocampal formation and claustrum, which are regions known to contain 5-CT insensitive 5-HT1 or non 5-HT1A/1B/IC/ID [3H]5-HT binding sites. Altogether, this ISHH study describes the distribution of mRNAs of recently cloned 5-HT1 receptors in rodent and primate brain and compares these results to the distribution of the heterogeneous population of 5-HT1 binding sites.

Breast cancer prognostic classification in the molecular era: the role of histological grade

Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. Current routine clinical management of breast cancer relies on the availability of robust clinical and pathological prognostic and predictive factors to support clinical and patient decision making in which potentially suitable treatment options are increasingly available. One of the best-established prognostic factors in breast cancer is histological grade, which represents the morphological assessment of tumor biological characteristics and has been shown to be able to generate important information related to the clinical behavior of breast cancers. Genome-wide microarray-based expression profiling studies have unraveled several characteristics of breast cancer biology and have provided further evidence that the biological features captured by histological grade are important in determining tumor behavior. Also, expression profiling studies have generated clinically useful data that have significantly improved our understanding of the biology of breast cancer, and these studies are undergoing evaluation as improved prognostic and predictive tools in clinical practice. Clinical acceptance of these molecular assays will require them to be more than expensive surrogates of established traditional factors such as histological grade. It is essential that they provide additional prognostic or predictive information above and beyond that offered by current parameters. Here, we present an analysis of the validity of histological grade as a prognostic factor and a consensus view on the significance of histological grade and its role in breast cancer classification and staging systems in this era of emerging clinical use of molecular classifiers.

Basal-like and triple-negative breast cancers: a critical review with an emphasis on the implications for pathologists and oncologists

Breast cancer is a heterogeneous disease encompassing a variety of entities with distinct morphological features and clinical behaviors. Although morphology is often associated with the pattern of molecular aberrations in breast cancers, it is also clear that tumors of the same histological type show remarkably different clinical behavior. This is particularly true for ‘basal-like cancer’, which is an entity defined using gene expression analysis. The purpose of this article was to review the current state of knowledge of basal-like breast cancers, to discuss the relationship between basal-like and triple-negative breast cancers, and to clarify practical implications of these diagnoses for pathologists and oncologists.

Muscarinic cholinergic receptor subtypes in the rat brain. I. Quantitative autoradiographic studies

The binding characteristics of N[3H]methylscopolamine ([3H]NMS) to slide-mounted tissue sections were studied using quantitative autoradiography. Binding of [3H]NMS was saturable, reversible and of high affinity (Kd = 0.26 nM). The inhibition of [3H]NMS binding produced by several muscarinic agonists and antagonists was analyzed in 29 discrete brain regions by constructing complete displacement curves. Comparison of IC50 values obtained both biochemically and by autoradiography demonstrated a very close agreement, supporting the validity of the autoradiographic approach. The competition curves for the agonists carbachol, oxotremorine and 2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion-h ydrobromide (RS 86) fitted to a two-site model, with comparable affinity values from region to region, although different proportions of high- and low-affinity sites were seen in the different areas studied. The distribution of high- and low-affinity sites was similar for the three agonists. Atropine showed monophasic curves presenting similar affinities in all regions studied. In contrast, pirenzepine differentiated between high- and low-affinity sites which showed a distribution opposite to that observed for the agonists. Gallamine, a ligand for a putative regulatory site in the muscarinic receptor, inhibited [3H]NMS binding in a biphasic manner. The calculated IC50 values for the gallamine high- and low-affinity sites did not vary from region to region and the distribution of these sites correlated well with that observed for the agonists. High-affinity pirenzepine sites (also called M1 sites) were localized mainly in forebrain areas, such as striatum, hippocampus and cortex, and their regional distribution correlated with that of the low-affinity sites for the agonists and gallamine. On the other hand, low-affinity sites for pirenzepine (named M2 sites) were mainly found in the brainstem and parts of the thalamus. A good correlation was found between pirenzepine low-affinity sites and agonist and gallamine high-affinity sites. The significance of these findings is discussed in relation to the known and possible effects of selective M1 and M2 centrally acting agents.

Combined Epidermal Growth Factor Receptor Targeting with the Tyrosine Kinase Inhibitor Gefitinib (ZD1839) and the Monoclonal Antibody Cetuximab (IMC-C225) Superiority Over Single-Agent Receptor Targeting

Purpose: The epidermal growth factor receptor (EGFR) is abnormally activated in cancer and two classes of anti-EGFR agents, monoclonal antibodies and low-molecular-weight tyrosine kinase inhibitors, have shown antitumor activity in patients. Because these two classes of antireceptor agents target the EGFR at different sites, we decided to explore whether the combined administration of gefitinib, a tyrosine kinase inhibitor, and cetuximab, a monoclonal antibody, had superior antitumor activity than either agent given alone. Experimental Design: We studied the effects of the combination of gefitinib and cetuximab in a panel of human cancer cell lines and in an EGFR-dependent human tumor xenograft model (A431). The effects of these two agents on EGFR signaling, proliferation, apoptosis, and vascularization were evaluated. In addition, we analyzed, with cDNA arrays, changes in gene expression profiles induced by both agents. Results: The combined treatment with gefitinib and cetuximab resulted in a synergistic effect on cell proliferation and in superior inhibition of EGFR-dependent signaling and induction of apoptosis. In a series of in vivo experiments, single-agent gefitinib or cetuximab resulted in transient complete tumor remission only at the highest doses. In contrast, suboptimal doses of gefitinib and cetuximab given together resulted in a complete and permanent regression of large tumors. In the combination-treated tumors, there was a superior inhibition of EGFR, mitogen-activated protein kinase, and Akt phosphorylation, as well as greater inhibition of cell proliferation and vascularization and enhanced apoptosis. Using cDNA arrays, we found 59 genes that were coregulated and 45 genes differentially regulated, including genes related to cell proliferation and differentiation, transcription, DNA synthesis and repair, angiogenesis, signaling molecules, cytoskeleton organization, and tumor invasion and metastasis. Conclusions: Our findings suggest both shared and complementary mechanisms of action with gefitinib and cetuximab and support combined EGFR targeting as a clinically exploitable strategy.

High affinity GABA receptors — Autoradiographic localization

The distribution of the high affinity gamma-aminobutyric acid (GABA) receptor labeled by [3H]muscimol, has been studied in the rat brain by light microscopic autoradiography. Receptors in slide-mounted tissue sections were labeled in vitro with [3H]muscimol. Most of the gray matter areas presented grain densities significantly higher than background or white matter areas. Wide variations in receptor densities were found between different brain areas and nuclei. Areas with very high grain densities are the granule cell layer of the cerebellum, external plexiform layer of the olfactory bulb and nuclei of the thalamus, such as the ventral nucleus, lateral nucleus and dorsal geniculate body. The molecular layer of the hippocampus and the external (I-IV) layers of the cortex are also rich in GABA receptors. The basal ganglia have moderate concentrations of receptors, while the pons, medulla and brainstem have only low concentrations of autoradiographic grains. These distributions are discussed in correlation with the known distribution of GABAergic terminals and the presence of inhibitory GABAergic mechanisms.