'Desperate house genes': the dramatic example of hypoxia.

BACKGROUND: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. METHODS: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). RESULTS: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. CONCLUSION: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

Prognostic value of prostate circulating cells detection in prostate cancer patients: a prospective study.

In clinically organ-confined prostate cancer patients, bloodstream tumour cell dissemination generally occurs, and may be enhanced by surgical prostate manipulation. To evaluate cancer-cell seeding impact upon patient recurrence-free survival, 155 patients were prospectively enrolled then followed. Here, 57 patients presented blood prostate cell shedding preoperatively and intraoperatively (group I). Of the 98 preoperatively negative patients, 53 (54%) remained negative (group II) and 45 (46%) became intraoperatively positive (group III). Median biological and clinical recurrence-free time was far shorter in group I (36.2 months, P<0.0001) than in group II (69.6 months) but did not significantly differ in group II and III (69.6 months vs 65.0). Such 5-year follow-up data show that preoperative circulating prostate cells are an independent prognosis factor of recurrence. Moreover, tumour handling induces cancer-cell seeding but surgical blood dissemination does not accelerate cancer evolution.

Computational approaches for the study of serotonin and its membrane transporter SERT: implications for drug design in neurological sciences.

Serotonin (5-hydroxytryptamine, 5-HT), a monoamine neurotransmitter of the central nervous and peripheral systems (CNS), plays a critical role in a wide variety of physiological and behavioral processes. In the serotonergic system, deregulation of the tightly controlled extracellular concentration of 5-HT appears to be at the origin of a host of metabolic and psychiatric disorders. A key step that regulates 5-HT external level is the re-uptake of 5-HT into cells by the 5-HT transporter (SERT), which is besides the target of numerous drugs interacting with the serotonergic system. Therapeutic strategies have mainly focused on the development of compounds that block the activity of SERT, for instance reuptake inhibitors (e.g. tricyclics, "selective" serotonin reuptake inhibitors) and in the past, specific substrate-type releasers (e.g. amphetamine and cocaine derivatives). Today, generation of new drugs targetting SERT with enhanced selectivity and reduced toxicity is one of the most challenging tasks in drug design. In this context, studies aiming at characterizing the physicochemical properties of 5-HT as well as the biological active conformation of SERT are a prerequisite to the design of new leads. However, the absence of a high-resolution 3D-structure for SERT has hampered the design of new transporter inhibitors. Using computational approaches, numerous efforts were made to shed light on the structure of 5-HT and its transporter. In this review, we compared several in silico methods dedicated to the modeling of 5-HT and SERT with an emphasis on i) quantum chemistry for study of 5-HT conformation and ii) ligand-based (QSAR and pharmacophore models) and transporter-based (homology models) approaches for studying SERT molecule. In addition, we discuss some methodological aspects of the computational work in connection with the construction of putative but reliable 3D structural models of SERT that may help to predict the mechanisms of neurotransmitter transport.

Definition of an uptake pharmacophore of the serotonin transporter through 3D-QSAR analysis.

The serotonergic system plays a critical role in a wide variety of physiological and behavioral processes. Dysregulation of the tightly controlled extracellular concentration of serotonin (5-hydroxytryptamine, 5-HT) appears to be at the origin of a host of metabolic and psychiatric disorders. Since the plasma membrane 5-HT transporter (SERT) is the major protagonist in regulating extracellular 5-HT concentration, SERT is the target of most drugs interacting with the serotonergic system. Unfortunately, some of the drugs towards SERT (e.g. amphetamine derivatives) interfere with cell homeostasis leading to cell toxicity. Developing new SERT ligands devoid of any side-effect represents a major priority in the treatment of 5-HT-associated pathologies. Here, we report structure-activity relationships (SAR) and three-dimensional QSAR (3D-QSAR) studies of a library of 121 compounds including 5-HT analogs, harmanes, benzothiazoles, indanones, amphetamine derivatives and substrate-type 5-HT releasers, with the goal of identifying the structural determinants crucial for SERT uptake. In the absence of data about the bioactive form of 5-HT, conformational analysis of 5-HT was performed using quantum chemistry calculations. This led to three 5-HT stable conformers with anti, -gauche and +gauche side-chain conformation. These conformers, used as templates for superimposition with all the library compounds, enabled the design of a reliable 6-points pharmacophore representative of SERT uptake activity. Molecular dynamics (MD) simulations performed with compounds that are efficiently, moderately, poorly or not transported by SERT allowed to assess the validity of our pharmacophore. Altogether, our data provide for the first time a reliable pharmacophore of SERT uptake activity, which may help to the design of new drugs targeting SERT.

Histological, immunohistochemical, ultrastructural and biochemical study of human gastric composite tumor: expression of the serotonin-2B receptor by the neuroendocrine component.

We report a case of a human gastric composite tumor occurring seven years after a partial gastrectomy for a low grade B cell MALT lymphoma. Histological examination of the tumor revealed two intimately intermingled components: 1. A moderately to poorly differentiated tubulo-acinar adenocarcinoma with signet-ring cells; and 2. Isolated or clustered small neuroendocrine cells without atypia expressing chromogranin A, somatostatin and/or glucagon, serotonin (5-HT) and, the 5-HT2B receptors. In addition to immunohistochemical detection, the presence of 5-HT2B receptors was shown pharmacologically through [125I]-DOI binding. Since 5-HT2B receptors have been demonstrated to have autocrine functions and, mitogenic and transforming properties, these results suggest a role of 5-HT in neuroendocrine malignant transformation. On the other hand, the expression of somatostatin and the detection by reverse transcriptase polymerase chain reaction (RT-PCR) of somatostatin receptor subtypes 2, 3, and 5, which have been shown to be involved in tumor regression, might account for the long evolution of this case (> 5 yr). This case illustrates the importance of local humoral modulation in tumor growth. Moreover, ultrastructural results favor a unique origin of the tumor cells from one amphicrine cell type.

Ablation of serotonin 5-HT(2B) receptors in mice leads to abnormal cardiac structure and function.

BACKGROUND: Identification of factors regulating myocardial structure and function is important to understand the pathogenesis of heart disease. Because little is known about the molecular mechanism of cardiac functions triggered by serotonin, the link between downstream signaling circuitry of its receptors and the heart physiology is of widespread interest. None of the serotonin receptor (5-HT(1A), 5-HT(1B), or 5-HT(2C)) disruptions in mice have resulted in cardiovascular defects. In this study, we examined 5-HT(2B) receptor-mutant mice to assess the putative role of serotonin in heart structure and function. METHODS AND RESULTS: We have generated G(q)-coupled 5-HT(2B) receptor-null mice by homologous recombination. Surviving 5-HT(2B) receptor-mutant mice exhibit cardiomyopathy with a loss of ventricular mass due to a reduction in number and size of cardiomyocytes. This phenotype is intrinsic to cardiac myocytes. 5-HT(2B) receptor-mutant ventricles exhibit dilation and abnormal organization of contractile elements, including Z-stripe enlargement and N-cadherin downregulation. Echocardiography and ECG both confirm the presence of left ventricular dilatation and decreased systolic function in the adult 5-HT(2B) receptor-mutant mice. CONCLUSIONS: Mutation of 5-HT(2B) receptor leads to a cardiomyopathy without hypertrophy and a disruption of intercalated disks. 5-HT(2B) receptor is required for cytoskeleton assembly to membrane structures by its regulation of N-cadherin expression. These results constitute, for the first time, strong genetic evidence that serotonin, via the 5-HT(2B) receptor, regulates cardiac structure and function.

Molecular organization of the human serotonin transporter at the air/water interface.

The serotonin transporter (SERT) is the target of several important antidepressant and psychostimulant drugs. It has been shown that under defined conditions, the transporter spread at the air/water interface was able to bind its specific ligands. In this paper, the interfacial organization of the protein has been assessed from dynamic surface pressure and ellipsometric measurements. For areas comprising between 10,400 and 7,100 A(2)/molecule, ellipsometric measurements reveal an important change in the thickness of the SERT film. This change was attributed to the reorientation of the transporter molecules from a horizontal to their natural predictive transmembrane orientation. The thickness of the SERT film at 7,100 A(2)/molecule was found to be approximately equal to 84 A and coincided well with the theoretical value estimated from the calculations based on the dimensions of alpha-helices containing membrane proteins. These data suggest that the three-dimensional arrangement of the SERT may be represented as a box with lengths d(z)=83--85 A and d(y) or d(x)=41--47 A.

[Biochemical mechanisms in the physiopathology of migraine]. / Mécanismes biochimiques dans la physiopathologie migraineuse.

Migraine is one of the few pathologies which gave rise to a tremendous number of physiopathological hypotheses. The variability of its clinical features and of the crisis initiating triggers, together with the numerous functional and/or biological abnormalities reported in migrainous patients, led to multiple 'theories' about migraine. For instance, migraine attacks may be associated with modifications of cerebral blood flow, and/or alterations at the cellular (neuronal and peripheral: platelets, mast cells, etc.) and subcellular (mainly mitochondrial) levels leading to variations of parameters such as serotonin, vasoactive neuropeptides, histamine, nitric oxide, neuroactive amino acids, etc. However, these modifications are mainly related to migraine attacks but not to migrainous patients. These emphasize how important is the distinction between the crisis mechanism(s) and the determinism of migraine illness. Despite the absence of any true animal model of migraine attack, the obtention, through the activation of the trigemino-vascular complex, of an experimental meningeal neurogenic inflammation was a clear breakthrough for the understanding of the migraine attack. Concerning the determinism of migraine, its familial characteristic has been known for a long time, but genetic studies started only recently. Despite some important contributions, the respective roles of genetic and environmental factors, as well as the transmission mode of migraine, remain largely to be determined. Practically, these genetic data, which really concern only a very peculiar form of migraine--the familial hemiplegic one--do not have presently any diagnostic or therapeutic application.

Identification of three novel mutations (E196K, V203I, E211Q) in the prion protein gene (PRNP) in inherited prion diseases with Creutzfeldt-Jakob disease phenotype.

Inherited prion diseases are characterized by mutations in the PRNP gene encoding the prion protein (PrP). As the other sporadic or infectious prion disease forms, they are almost all characterized by the accumulation in the brain of an abnormal misfolded form of the patient's PrP. Brain extracts can often transmit the disease once inoculated in a recipient animal. Inherited prion diseases with Creutzfeldt-Jakob disease (CJD) phenotype are autosomal forms, although sporadic cases have been reported. We report three novel mutations of the PRNP gene in unrelated patients with clinical and histopathologic features of CJD. The three mutations were missense: c635G>A (E196K), c656G>A (V203I) and c680G>C (E211Q). Familial history of neurologic disorders was evidenced for patients carrying the E196K and E211Q mutations. E196K would be predicted to have more severe effects on protein stability than V203I and E211Q. These mutations expand the spectrum of mutations in PRNP and reduce the proportion of CJD patients in whom genetic alterations have not been found.

Ligand interaction with the purified serotonin transporter in solution and at the air/water interface.

The purified serotonin transporter (SERT) was spread at the air/water interface and the effects both of its surface density and of the temperature on its interfacial behavior were studied. The recorded isotherms evidenced the existence of a stable monolayer undergoing a lengthy rearrangement. SERT/ligand interactions appeared to be dependent on the nature of the studied molecules. Whereas an unrelated drug (chlorcyclizine) did not bind to the spread SERT, it interacted with its specific ligands. Compared to heterocyclic drugs, for which binding appeared to be concentration-dependent, a 'two-site' mechanism was evidenced for pinoline and imipramine.

PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor.

Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5-HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK(-) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.

Decreased serotonin transporter binding in unaffected relatives of manic depressive patients.

BACKGROUND: There are numerous reports of decreased binding to platelet serotonin transporter (5-HTT) in depression, suggesting that it might be considered a trait marker of depression. To further investigate whether reduced 5-HTT function could be an endophenotype in manic depressive illness, we looked for abnormalities of platelet 5-HTT among subjects who are potential carriers of genetic vulnerability to manic depressive illness (MDI). METHODS: Blood samples were obtained from 20 unaffected relatives from families with at least two individuals with bipolar disorder and from 19 control participants. Plasma 5-HIAA, platelet 5-HT, and [3H] imipramine binding were measured. RESULTS: Unaffected relatives manifested lower platelet 5-HTT function than control participants as revealed both by reduced number and diminished affinity of imipramine binding sites and diminished platelet 5-HT content. CONCLUSIONS: These preliminary results suggest that reduced 5-HTT function could be considered a trait marker or an endophenotype in MDI.

Cross-talk between 5-hydroxytryptamine receptors in a serotonergic cell line. Involvement of arachidonic acid metabolism.

The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis.