The apoptotic members CD95, BclxL, and Bcl-2 cooperate to promote cell migration by inducing Ca(2+) flux from the endoplasmic reticulum to mitochondria.

Metalloprotease-processed CD95L (cl-CD95L) is a soluble cytokine that implements a PI3K/Ca(2+) signaling pathway in triple-negative breast cancer (TNBC) cells. Accordingly, high levels of cl-CD95L in TNBC women correlate with poor prognosis, and administration of this ligand in an orthotopic xenograft mouse model accelerates the metastatic dissemination of TNBC cells. The molecular mechanism underlying CD95-mediated cell migration remains unknown. Here, we present genetic and pharmacologic evidence that the anti-apoptotic molecules BclxL and Bcl-2 and the pro-apoptotic factors BAD and BID cooperate to promote migration of TNBC cells stimulated with cl-CD95L. BclxL was distributed in both endoplasmic reticulum (ER) and mitochondrion membranes. The mitochondrion-localized isoform promoted cell migration by interacting with voltage-dependent anion channel 1 to orchestrate Ca(2+) transfer from the ER to mitochondria in a BH3-dependent manner. Mitochondrial Ca(2+) uniporter contributed to this flux, which favored ATP production and cell migration. In conclusion, this study reveals a novel molecular mechanism controlled by BclxL to promote cancer cell migration and supports the use of BH3 mimetics as therapeutic options not only to kill tumor cells but also to prevent metastatic dissemination in TNBCs.

Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins.

Alteration of the stability of Bag-1 protein in the control of olfactory neuronal apoptosis.

Normal apoptosis occurs continuously in the olfactory neuroepithelium of adult vertebrates, making it a useful model for studying neuronal apoptosis. Here we demonstrate that overexpression of the anti-apoptotic Bag-1 gene in olfactory neuronal cells confers a strong resistance to apoptosis. Conversely decreased levels of Bag-1 were found to precede a massive wave of olfactory neuronal apoptosis triggered by synaptic target ablation. We show that the decrease is brought about by ubiquitination and subsequent degradation of the Bag-1 protein. The ring finger protein Siah-2 is a likely candidate for the ubiquitination reaction since Siah-2 mRNA accumulated in lesioned olfactory neuroepithelium and overexpression of Siah-2 stimulated Bag-1 ubiquitination and degradation in transient expression assays. These results together identify destabilization of Bag-1 as a necessary step in olfactory neuronal apoptosis.

Postnatal development of the tyrosine hydroxylase-containing cell population within the rat locus coeruleus: topological organization andphenotypic plasticity.

The cellular phenotypic characteristics of tyrosine hydroxylase (TH) expression have been studied within the rat locus coeruleus (LC) during postnatal development at six different stages: postnatal day 4 (PND4), PND10, PND14, PND21, PND30, and PND42. Coronal brain sections were selected at intervals of 80 microns along the caudorostral extent of the LC and processed for TH immunohistochemistry. At each anatomical level we (1) reconstructed the mean space of the LC delineated by the TH positive cell bodies, (2) enumerated the mean number of these cell bodies, and (3) determined the mean volume circumscribed by these cell bodies and their density. The topological study revealed a steady remodeling of the structure until the third week, with a progressive reducing of a ventral cellular group in the anterior LC, which was no longer observable at PND21, concomitant to the stretch of the structure toward its caudal limit. We have noted invariant and variant cellular phenotypic characteristics of TH expression. At any stage, the LC could be separated into a posterior and an anterior subregion and its total volume remained quite stable during the studied period. At PND14 and PND21, we observed a transient 33% increase in the total number of TH positive perikarya as compared to PND42. Conjoint analysis of the topological reconstruction and the density of TH positive cells suggested there were three distinct and precisely localized subsets of "quiescent" neurons. TH gene expression in these cells would have lowered between PND14 and PND21 inside two subsets and between PND21 and PND30 inside the last one. So topologically defined populations of cells could be involved in specific functions. If they have not definitively lost their TH expression capacity, they could contribute to increasing TH levels in LC occurring in response to physiological perturbations or pharmacological treatments.

RU24722 induces spatially organized phenotypic plasticity in the locus coeruleus.

The plasticity of tyrosine hydroxylase (TH) expression in rat locus coeruleus (LC) was evaluated after RU24722 TH induction using, as a new parameter of characterization, the quantitative topology of LC defined by TH-positive cells. This new phenotype was spatially organized into cell subpopulations in the medial LC, dorsal and ventro-lateral to the initial perikaryal space. Reserpine and parachlorophenylalanine, which elicited a similar increase in the TH content, failed to induce a significant change in the number of TH-expressing cells. Activation of TH expression is not sufficient to reveal the existence of such a plasticity and some original but still unknown mechanism(s) of control of TH expression is affected by RU24722.

Relationship between tyrosine hydroxylase content and noradrenergic cell reactivity to piperoxane: an in vivo voltammetric approach in the rat locus coeruleus.

A previous electrochemical study showed that the increase in the tyrosine hydroxylase (TH) content of the locus coeruleus (LC) produced by RU24722 administration was associated with a relative decrease in the catecholaminergic metabolic reactivity of this nucleus to a hypotensive stimulus. Since alpha 2 receptors participate in the regulation of the activity of both LC neurons and TH, the aim of the present work was to evaluate the possible involvement of the autoinhibition mediated by alpha 2 autoreceptors in the inverse relationship between the reactivity of the LC and its TH content. Our study was divided into two successive steps: (i) the electrochemical measurement of the in vivo metabolic activation of LC cells in response to alpha 2-adrenergic receptor blockade, and (ii) the evaluation of the quantity of TH every 100 microns along the caudorostral axis in each recorded LC. The capacity of TH protein to be activated was evaluated by the measurement, using differential normal pulse voltammetry, of the in vivo variations of the extracellular 3,4-dihydroxyphenylacetic acid concentrations in response to six cumulated doses of the alpha 2-antagonist piperoxane. The corresponding dose-response curves, determined in control- and RU24722-treated rats, were expressed as a function of the quantity of TH contained either in the whole recorded LC or in the 100 microns-wide coronal interval surrounding the recording site. It was established that the slopes of the dose-response curves were significantly (P < 0.01) and inversely related to the quantity of TH at the level of the recording site. This result suggests that the negative control of the catecholaminergic metabolic reactivity in a restricted area of the LC could be directly or indirectly dependent on the level of expression of TH protein in this particular area.

Somatotopic organization of tyrosine hydroxylase expression in the rat locus coeruleus: long term effect of RU24722.

Tyrosine hydroxylase (TH) tissue concentration was determined by immunostaining of tissue sections directly transferred onto nitrocellulose membranes in the restricted region of the noradrenergic perikarya of the locus coeruleus (LC) along its postero-anterior axis. TH containing cells were systematically counted on adjacent post fixed sections stained by immunohistochemistry. The absolute quantity of TH was estimated in each section and was found to be linearly related to the number of TH immuno-positive cells found in the adjacent section. The ratio between these two parameters was thus used as an index of the cellular concentration of TH in noradrenergic cells. In the LC of control rats, the TH cellular concentration was lower (-39%) in the anterior than in the posterior half of the structure. Three days after an injection of 20 mg/kg of RU24722, an eburnamine derivative known to increase the quantity of TH in the LC, increases in quantities of TH were found in both portions of the LC. Moreover in the posterior LC the increase in the amount of TH resulted from a significant increase in the number of TH-immunopositive cells. In the anterior part, however, it was primarily the result of a significant increase in TH cellular concentration. Throughout the LC there was an increase in the cellular concentration of TH which was inversely proportional to the concentrations found in control animals. TH mRNA content was measured by a quantitative in situ hybridization in sections of both the posterior and anterior LC one day after a single injection of RU24722 at the same dose. The quantity of TH mRNA was significantly increased in both parts. The number of TH mRNA-expressing neurons also increased, especially in the anterior LC. Thus the effects at the level of TH protein and TH mRNA were strikingly parallel though increase in TH protein occurred later than the increase in the TH mRNA. These results suggest that in the rat LC: (1) there is a significant population of 'sleeping cells' in which TH expression is either inactivated or, at a low level of activation; (2) TH cellular concentration could exert a retrocontrol on its own expression in cells of the LC that contained TH and (3) TH expression appears to be regulated by different selective mechanisms in these two different subpopulations of noradrenergic cells within the LC.

Variation of tryptophan-5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration. II. Anatomical distribution of the tryptophan-5-hydroxylase protein and regional variation of its turnover rate.

Distribution of tryptophan-5-hydroxylase (TpOH)-containing cells and TpOH protein tissue concentrations were evaluated in the nucleus raphe dorsalis (NRD) of rat brain by immunocytochemistry and direct transfer onto nitrocellulose filters of unfixed adjacent brain sections. This work has demonstrated that: (1) the direct transfer onto nitrocellulose filters could be easily used for the quantitative analysis of TpOH protein distribution; (2) the origin of the TpOH in this brain nucleus was preferentially cellular; (3) classical subdivisions, qualitatively defined from morphometric and topographic observations could be precisely described in terms of cellular density, tissue and cellular concentrations and turnover of TpOH protein. Such differences could imply a physiological control of TpOH gene expression in the serotoninergic neurons.