Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo.

BACKGROUND: The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear. METHODS/RESULTS: In the present study, we showed by qRT-PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton. CONCLUSION: Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.

Differential expression and redox proteomics analyses of an Alzheimer disease transgenic mouse model: effects of the amyloid-ß peptide of amyloid precursor protein.

Among the pathological factors known to be associated with Alzheimer disease (AD), oxidative stress induced by the amyloid-ß peptide (Aß) has been demonstrated to play a key role in human brain and animal models of AD. Recently, we reported elevated levels of oxidative damage in the brain of a transgenic (Tg) AD mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) [PDAPP mice, line J20], as evidenced by increased levels of protein carbonyls, 3-nitrotyrosine, and protein-bound 4-hydroxy-2-nonenal. This oxidative damage was dependent on the methionine 35 residue within the Aß peptide. Further insight into the molecular pathways affected in this Tg model of AD may be gained with discovery-based proteomics studies; therefore, two-dimensional gel-based expression proteomics was performed to compare differences in brain protein levels of J20 Tg mice with non-transgenic (NTg) littermate controls. Based on our studies, we identified six proteins that had significantly increased levels in J20 Tg relative to NTg mice: calcineurin subunit B type 1, ρ GDP-dissociation inhibitor 1, T-complex protein 1 subunit α A, α-enolase, peptidyl-prolyl cis-trans isomerase (Pin-1), and ATP synthase subunit α mitochondrial. Several of these proteins have previously been implicated in in vitro and in vivo models and subjects with AD. Additionally, using redox proteomics analyses we identified two oxidatively-modified proteins: phosphatidylethanolamine-binding protein 1 and Pin-1 with decreased levels of protein 3-nitrotyrosine in J20 Tg mice relative to NTg. Western blotting and immunoprecipitation analyses were used to validate proteomics results. Overall, these studies provide information about changes in the brain proteome as a result of Aß deposition and clues with which to further direct studies on elucidating AD pathogenesis.

Netrin-1 interacts with amyloid precursor protein and regulates amyloid-beta production.

The beta-amyloid precursor protein (APP) is an orphan transmembrane receptor whose physiological role is largely unknown. APP is cleaved by proteases generating amyloid-beta (Abeta) peptide, the main component of the amyloid plaques that are associated with Alzheimer's disease. Here, we show that APP binds netrin-1, a multifunctional guidance and trophic factor. Netrin-1 binding modulates APP signaling triggering APP intracellular domain (AICD)-dependent gene transcription. Furthermore, netrin-1 binding suppresses Abeta peptide production in brain slices from Alzheimer model transgenic mice. In this mouse model, decreased netrin-1 expression is associated with increased Abeta concentration, thus supporting netrin-1 as a key regulator of Abeta production. Finally, we show that netrin-1 brain administration in Alzheimer model transgenic mice may be associated with an amelioration of the Alzheimer's phenotype.

A novel paraptosis pathway involving LEI/L-DNaseII for EGF-induced cell death in somato-lactotrope pituitary cells.

We have recently reported that EGF triggers an original form of cell death in pituitary cell line (GH4C1) with a phenotype sharing some characteristics of both apoptosis (internucleosomal DNA fragmentation) and paraptosis (caspase-independence and cytoplasmic vacuolization). However, the endonuclease involved in EGF-induced DNA fragmentation has not been assessed so far. In the present work we therefore further explored the putative paraptosis involvement in EGF-induced cell death and asked whether L-DNaseII might be involved. Indeed, this endonuclease is known to mediate internucleosomal DNA fragmentation in caspase independent manner. Our Western blot, immunocytochemistry and enzymatic measurement assays show that EGF triggers a cleavage of Leukocyte Elastase Inhibitor (LEI) precursor into L-DNaseII, its subsequent enzymatic activation and nuclear translocation thus pointing to the involvement of this endonuclease pathway in caspase-independent DNA fragmentation. In addition, EGF-induced cell death can be blocked by paraptosis inhibitor AIP-1/Alix, but not with its anti-apoptotic C-terminal fragment (Alix-CT). Altogether these data suggest that EGF-induced cell death defines a novel, L-DNaseII-mediated form of paraptosis.

Characterization of neural stem cells in the dorsal vagal complex of adult rat by in vivo proliferation labeling and in vitro neurosphere assay.

The dorsal vagal complex, located in the brainstem, is the major integrative center of the autonomic nervous system. By combining in vivo bromodeoxyuridine incorporation and phenotypic immunolabeling, we have previously reported that neurogenesis occurs in the adult rat dorsal vagal complex [Bauer S, Hay M, Amilhon B, Jean A, Moyse E (2005) In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem. Neuroscience 130:75-90.]. In the present study we asked whether adult dorsal vagal complex contains proliferative and/or neural stem cells. Using Ki-67 immunolabeling and cyclin D1 Western blot, we showed intrinsic cell proliferation in the dorsal vagal complex and its stimulation by vagotomy. Detailed time-course analysis revealed that vagotomy-induced proliferation in the dorsal vagal complex peaked three days after lesion. In order to directly assess the presence of intrinsic stem cells, primary cell cultures from adult rat dorsal vagal complex were performed in the presence of epidermal growth factor and basic fibroblast growth factor (neurosphere assay). A discrete subpopulation of dorsal vagal complex cells proliferated as neurospheres, self-renewed when passaged, and differentiated into neurons, astrocytes and oligodendrocytes. Proliferation and neuron-differentiating potentials of dorsal vagal complex neurospheres were both lower than those of subventricular zone neurospheres from the same rats. The relationship between in vitro neurosphere-forming cells of dorsal vagal complex and in vivo dorsal vagal complex neurogenesis is discussed and remains to be directly addressed. The present data demonstrate the occurrence of neural stem cells in the dorsal vagal complex of adult rat brain.

Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone.

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) where dopamine D3 receptors are expressed. Here, we demonstrate that addition of 1 microm apomorphine increases cell numbers in post-natal SVZ cell cultures. This effect was prevented by a co-treatment with haloperidol, sulpiride or U-99194A, a D3-preferring antagonist, and mimicked by the dopamine D3 receptor selective agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). EC50 values were 4.04 +/- 1.54 nm for apomorphine and 0.63 +/- 0.13 nm for 7-OH-DPAT, which fits the pharmacological profile of the D3 receptor. D3 receptors were detected in SVZ cells by RT-PCR and immunocytochemistry. D3 receptors were expressed in numerous beta-III tubulin immunopositive cells. The fraction of apoptotic nuclei remained unchanged following apomorphine treatment, thus ruling out any possible effect on cell survival. In contrast, proliferation was increased as both the proportion of nuclei incorporating bromo-deoxyuridine and the expression of the cell division marker cyclin D1 were enhanced. These findings provide support for a regulatory role of dopamine over cellular dynamics in post-natal SVZ.

Novel modalities of somatostatin actions.

The first part of this contribution reviews the current knowledge about endocrine and neuromodulatory actions of somatostatin. These biological actions are exerted according to endocrine, paracrine and autocrine modes of action and involve five distinct types of membrane receptors belonging to the 'super-family' of G-protein-coupled receptors. A new concept concerning a juxtacrine mode of action has recently been introduced to refer to the intervention of cytokines and growth factors in direct, cell-to-cell communication. The evidence in favor of juxtacrine actions of somatostatin will be presented in the second part of this review and illustrated by our own results on macrophage-lymphocyte T interactions in the immune system and spermatogonia-Sertoli cell interactions in mammalian testis. Another phenomenon such as ligand-induced somatostatin receptor homo- and hetero-dimerization resulting in 'poly'-receptors (with characteristics different from those of each of the two receptors forming the complex) is also at the origin of a novel mode of somatostatin action. The latter will be illustrated by the data obtained on human pituitary adenomas with somatostatin analogs specific for either 'poly'-receptor or relevant individual receptors. The arguments in favor of the analogous mode of actions among different families of chemical messengers such as peptides, cytokines and growth factors are discussed in the concluding section. The emerging unifying concepts on such functional analogies might provide a useful basis for the development of synthetic analogs not only for bioactive peptides but also for other types of chemical messengers.