Identification of stromal proteins overexpressed in nodular sclerosis Hodgkin lymphoma

Hodgkin lymphoma (HL) represents a category of lymphoid neoplasms with unique features, notably the usual scarcity of tumour cells in involved tissues. The most common subtype of classical HL, nodular sclerosis HL, characteristically comprises abundant fibrous tissue stroma. Little information is available about the protein composition of the stromal environment from HL. Moreover, the identification of valid protein targets, specifically and abundantly expressed in HL, would be of utmost importance for targeted therapies and imaging, yet the biomarkers must necessarily be accessible from the bloodstream. To characterize HL stroma and to identify potentially accessible proteins, we used a chemical proteomic approach, consisting in the labelling of accessible proteins and their subsequent purification and identification by mass spectrometry. We performed an analysis of potentially accessible proteins in lymph node biopsies from HL and reactive lymphoid tissues, and in total, more than 1400 proteins were identified in 7 samples. We have identified several extracellular matrix proteins overexpressed in HL, such as versican, fibulin-1, periostin, and other proteins such as S100-A8. These proteins were validated by immunohistochemistry on a larger series of biopsy samples, and bear the potential to become targets for antibody-based anti-cancer therapies

Expression et rôle fonctionnel de la troponine C dans l'activité contractile, en conditions normales et après un épisode d'hypodynamie - hypokinésie

Ce travail a pour objectif l'étude des changements d'expression d'une protéine régulatrice de la contraction musculaire, la troponine c (tnc), et des transformations fonctionnelles corrélatives, sur des muscles à fonction posturale après une période d'hypodynamie-hypokinésie (hh) de 14 jours (microgravité simulée) ou de microgravité réelle. L'expression relative des deux isoformes de tnc (lente tncs, ou rapide tncf), sur muscles entiers et sur fibres isolées, a été établie par immunodétection. Les propriétés fonctionnelles, sur des fibres isolées et pelées, ont été analysées à partir des propriétés d'activation par le ca 2 + et le sr 2 +, en absence et en présence d'un outil pharmacologique, le bepridil. Ce dernier cible spécifiquement le comportement de la tnc. Sur le soleus contrôle de rat, la tncs est exprimée de façon majoritaire (88%) et se répartit dans des fibres n'exprimant que cette isoforme (fibres lentes l, 74%), ou des fibres hybrides coexprimant tncs et tncf (hybrides lentes hl : 10%, ou hybrides rapides hr : 16%). Aucune fibre exprimant uniquement la tncf n'a été répertoriée.Apres hh, on observe au niveau du soleus atrophié une augmentation de l'expression de la tncf, due a une augmentation de la proportion de fibres hr (16 a 45%) au détriment des fibres l (45%). Ces fibres hr possèdent les mêmes caractéristiques d'activation que les fibres contrôles correspondantes, liées au type de tnc prédominante. En revanche, les fibres l et hl acquièrent après hh des caractéristiques d'activation spécifiques, non imputables a un changement du fonctionnement de la tnc. Les données obtenues après microgravité réelle sur un autre muscle extenseur lent (triceps de singe) confirment ces résultats. L'hh induit par conséquent un changement d'expression de la tnc rendant compte des transformations fonctionnelles des fibres musculaires. Nos résultats montrent cependant que d'autres protéines régulatrices contribuent a ces modifications, notamment sur les fibres demeurées lentes.LILLE1-BU (590092102) / SudocSudocFranceF

Procédé de criblage in-vitro de marqueurs biologiques accessibles dans des tissus pathologiques

publication date: 2010-07-07; filing date: 2006-12-13An in-vitro method for screening accessible biological markers in pathologic tissue

Characterization of an antibody panel for immunohistochemical analysis of canine muscle cells

Immunohistochemistry is an indispensable tool in the assessment and characterization of lineage-specific differentiation of grafted cells in cell-based-therapy. This strategy is under investigation for the treatment of many muscle disorders and different animals such as dogs are used as models to study the tissue regeneration. The aim of the present study was to characterize an antibody panel for the analysis of canine muscle cells, useful in routinely processed formalin-fixed paraffin-embedded tissues. Overall, 12 antibodies (8 mouse monoclonal and 4 goat polyclonal), validated for use on human tissues tested for cross-reactivity on canine smooth muscle (bladder, intestine, and uterus), skeletal muscle and heart. Specific staining was achieved with eight antibodies, of which six were cytoplasmic markers (desmin, HDAC8, MHC, SMA, Troponin I and Troponin T) and two were cardiac nuclear markers (GATA-4 and Nkx-2.5). This antibody panel may be useful not only for the evaluation of cell-based therapies in muscle disorders, but also for the evaluation of canine soft tissue neoplasms in veterinary pathology

Overexpression of CD9 in human breast cancer cells promotes the development of bone metastases.

BACKGROUND: Bone is a preferred target for circulating metastatic breast cancer cells. We found that the CD9 protein was up-regulated in the B02 osteotropic cell line, derived from the aggressive parental MDA-MB-231 breast cancer cell line. Here, we investigated the putative relationship between CD9 expression and the osteotropic phenotype. MATERIALS AND METHODS: Overexpression of CD9 was analyzed by immunoblotting in different cell lines. Immunohistochemistry was used to assess CD9 expression in primary tumors and metastatic lesions. In vivo experiments were conducted in mice using a monoclonal antibody against CD9. RESULTS: CD9 overexpression was confirmed in osteotropic cells. CD9 was significantly overexpressed in bone metastases versus primary tumors and visceral metastatic lesions. Finally, in vivo experiments showed that an antibody against CD9 delays homing of B02 cells in bone marrow, slowing down bone destruction. CONCLUSION: Our study reveals a potential implication of CD9 in the formation of bony metastases from breast cancer cells

IP(3)R3 silencing induced actin cytoskeletal reorganization through ARHGAP18/RhoA/mDia1/FAK pathway in breast cancer cell lines

International audienceCell morphology is altered in the migration process, and the underlying cytoskeleton remodeling is highly dependent of intracellular Ca2+ concentration. Many calcium channels are known to be involved in migration. Inositol 1,4,5-trisphosphate receptor (IP3R) was demonstrated to be implicated in breast cancer cells migration, but its involvement in morphological changes during the migration process remains unclear. In the present work, we showed that IP(3)R3 expression was correlated to cell morphology. IP(3)R3 silencing induced rounding shape and decreased adhesion in invasive breast cancer cell lines. Moreover, IP(3)R3 silencing decreased ARHGAP18 expression, RhoA activity, Cdc42 expression and (Y861)FAK phosphorylation. Interestingly, IP(3)R3 was able to regulate profilin remodeling, without inducing any myosin II reorganization. IP3R3 silencing revealed an oscillatory calcium signature, with a predominant oscillating profile occurring in early wound repair. To summarize, we demonstrated that IP3R3 is able to modulate intracellular Ca2+ availability and to coordinate the remodeling of profilin cytoskeleton organization through the ARHGAP18/RhoA/mDia1/PAK pathway

Cell Membrane Proteomic Analysis Identifies Proteins Differentially Expressed in Osteotropic Human Breast Cancer Cells12

Metastatic breast cancer cells are characterized by their high propensity to colonize the skeleton and form bone metastases, causing major morbidity and mortality. Identifying key proteins involved in the osteotropic phenotype would represent a major step toward the development of both new prognostic markers and new effective therapies. Cell surface proteins differentially expressed in cancer cells are preferred potential targets for antibody-based targeted therapies. In this study, using cell surface biotinylation and a mass spectrometric approach, we have compared the profile of accessible cell surface proteins between the human breast cancer cell line MDA-MB-231 and its highly osteotropic B02 subclone. This strategy allowed the identification of several proteins either up- or downregulated in the osteotropic cell line, and differential protein expressions were validated using antibody-based techniques. Class I HLAs were down-regulated in the bone metastatic variant, whereas αvβ3 integrins, among others, were consistently up-regulated in this latter cell line. These results show that comprehensive profiling of the cell surface proteome of mother cancerous cell lines and derived organ-specific metastatic cell lines provides an effective approach for the identification of potential accessible marker proteins for both prognosis and antibody-based targeted therapies

A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes

Expression and functional properties of four slow skeletal troponin T isoforms in rat muscles

We investigated the expression and functional properties of slow skeletal troponin T(sTnT) isoforms in rat skeletal muscles. Four sTnT cDNAs were cloned from the slow soleus muscle. Three isoforms were found to be similar to sTnT1, sTnT2, and sTnT3 isoforms described in mouse muscles. A new rat isoform, with a molecular weight slightly higher than that of sTnT3, was discovered. This fourth isoform had never been detected previously in any skeletal muscle and was therefore called sTnTx. From both expression pattern and functional measurements, it appears that sTnT isoforms can be separated into two classes, high-molecular-weight ( sTnT1, sTnT2) and low-molecular-weight ( sTnTx, sTnT3) isoforms. By comparison to the apparent migration pattern of the four recombinant sTnT isoforms, the newly described low-molecular-weight sTnTx isoform appeared predominantly and typically expressed in fast skeletal muscles, whereas the higher-molecular-weight isoforms were more abundant in slow soleus muscle. The relative proportion of the sTnT isoforms in the soleus was not modified after exposure to hindlimb unloading (HU), known to induce a functional atrophy and a slow-to-fast isoform transition of several myofibrillar proteins. Functional data gathered from replacement of endogenous troponin complexes in skinned muscle fibers showed that the sTnT isoforms modified the Ca2+ activation characteristics of single skeletal muscle fibers, with sTnT2 and sTnT1 conferring a similar increase in Ca2+ affinity higher than that caused by low-molecular-weight isoforms sTnTx and sTnT3. Thus we show for the first time the presence of sTnT in fast muscle fibers, and our data show that the changes in neuromuscular activity on HU are insufficient to alter the sTnT expression pattern.MICAD