Challenges in the stratification of breast tumors for tailored therapies.

Studying the molecular stratification of breast carcinoma is a real challenge considering the extreme heterogeneity of these tumors. Many patients are now treated following recommendation established at several NIH and St Gallen consensus conferences. However a significant fraction of these breast cancer patients do not need adjuvant chemotherapies while other patients receive inefficacious therapies. High density gene expression arrays have been designed to attempt to establish expression profiles that could be used as prognostic indicators or as predictive markers for response to treatment. This review is intended to discuss the potential value of these new indicators, but also the current weaknesses of these new genomic and bioinformatic approaches. The combined analysis of transcriptomic and genomic alteration data from relatively large numbers of well annotated tumor specimens may offer an opportunity to overcome the current difficulties in validating recently published non overlapping gene lists as prognostic or therapeutic indicators. There is also hope for identifying and deciphering signal transduction pathways driving tumor progression with newly developed algorithms and semi quantitative parameters obtained in simplified in vitro or in vivo models for specific transduction pathways.

Characterisation of serotonin transport mechanisms in rainbow trout peripheral blood lymphocytes: role in PHA-induced lymphoproliferation.

In this study, we investigated the serotonin transport mechanisms in rainbow trout (Oncorhynchus mykiss) peripheral blood Lymphocytes. We have observed that the transport of serotonin is a membrane transport process that have the properties of a secondary active transport system. The binding isotherm of [3H]-paroxetine, a serotonin transport blocker, demonstrated a high-affinity binding site with a positive type of cooperativity, Hill coefficient being higher than unity. Known specific inhibitors of the mammalian serotonin transporter significantly inhibited the uptake process in fish lymphocytes. In order to demonstrate the physiological relevance of the serotonin transporter in T-cell activation, we conducted experiments on lymphocytes activated or not by phytohemagglutinin (PHA), a T-cell mitogen. We have observed that addition of PHA for 24hrs, increased the Vmax but not the Km of this transporter. Serotonin uptake inhibitors diminished the PHA-activated proliferation of fish lymphocytes. The intracellular concentrations of cAMP were found to regulate the serotonin uptake and the PHA-stimulated proliferation as the agents known to augment cAMP stimulated serotonin uptake, and inhibited the lymphoproliferation. Inhibitory effects of increased cAMP on the proliferation were reversed by the addition of the nanomolar concentrations of 8-OH-DPAT, a 5-HT1A receptor agonist which is known to diminish the intracellular cAMP concentrations, suggesting that serotonin also regulates PHA-induced proliferation via 5-HT1A membrane receptors in an autocrine manner. These results all together demonstrate that fish lymphocytes possess an active serotonin transporter that is implicated in the proliferation of these immunocompetent cells.

5-Hydroxytryptamine-induced calcium-channel gating in rainbow trout (Oncorhynchus mykiss) peripheral blood lymphocytes.

The present study was conducted on peripheral blood lympho-cytes of rainbow trout (Oncorhynchus mykiss) to assess the role of 5-hydroxytryptamine (5-HT; 'serotonin') in calcium signalling. 5-HT-induced increases in intracellular free calcium concentrations, [Ca2+]i, and its action was mediated by 5-HT receptor subtype 3 (5-HT3), but not by 5-HT receptor subtype 1A (5-HT1A) or subtype 2 (5-HT2) in these cells. In Ca2+-containing medium (1 mM CaCl2), 5-HT and 2-methyl-5-HT (5-HT3 receptor agonist) induced increases in [Ca2+]i, whereas in Ca2+-free medium (0 Ca2+, 1 mM EGTA), these two agents failed to evoke increases in [Ca2+]i in these cells, demonstrating that 5-HT mobilizes Ca2+ from the extracellular environment. Furthermore, 5-HT-induced increases in [Ca2+]i are not contributed to by the intracellular endoplasmic reticulum (ER) pool, as thapsigargin, an agent that recruits Ca2+ from ER stores, had additive effects on 5-HT-induced [Ca2+]i responses in fish peripheral lymphocytes. 5-HT-induced increases in [Ca2+]i were mediated by 5-HT3 receptors via gating the calcium through L-type, but not N-type, calcium channels in trout lymphocytes.

Identification of lymphocyte 5-HT3 receptor subtype and its implication in fish T-cell proliferation.

In the present study, we identified the serotonergic receptor of type 3 (5-HT3) on the lymphocytes of a teleost fish, Oncorhynchus mykiss. In the pharmacological studies on the binding of [3H]serotonin to membrane receptor sites, 2-methyl-5-HT, an agonist of 5-HT3 receptors, displaced the binding of [3H]serotonin to fish lymphocytes, indicating the presence of 5-HT3 receptors on these cells. The known antagonists of the mammalian 5-HT3 receptor, ICS-205-930 and metoclopramide, failed to displace [3H]serotonin binding to lymphocytes during the period of association equilibrium (8 min); however, these antagonists progressively displaced [3H]serotonin binding from 10 to 40 min of incubation. These results suggest that fish 5-HT3 lymphocyte receptors may differ pharmacologically from mammalian receptors. As mammalian 5-HT3 receptors are coupled with Na+ inward movements, we undertook a study on Na+ influx by using SBFI/AM, a fluorescent probe. In SBFI/AM loaded fish lymphocytes, 2-methyl-5-HT leaked Na+ inward movements. Prior incubation of lymphocytes for 30 min in the presence of 5-HT3 antagonists, ICS-205-930, metoclopramide and MDL-72222, curtailed significantly the Na+ influx evoked by 2-methyl-5-HT, demonstrating that Na+ is leaked into fish lymphocytes via the 5-HT3 receptor-channel whose functioning is blocked by these antagonists. Furthermore, 2-methyl-5-HT exerted immunosuppressive effects in a dose dependent manner on fish T-lymphocytes stimulated by phytohaemagglutinin (PHA). Serotonin and 2-methyl-5-HT blocked the cell cycle progression of PHA-stimulated T-cells from G0/G1 to S phase. The immunosuppressive effects of 2-methyl-5-HT on T-cells were partially reversed by the antagonists, metoclopramide and ICS-205-930; however, the latter antagonist at high concentrations synergized with the immunosuppressive effects of 2-methyl-5-HT. These results demonstrate that the fish lymphocyte 5-HT3 receptor, which may be pharmacologically different from mammalian receptor subtype, is functionally implicated in fish T-cell proliferation.