[Place of radiotherapy in the treatment of rectal cancer in the elderly]. / Place de la radiothérapie dans le traitement du cancer du rectum chez le sujet âgé.

Rectal cancer is a common disease of the elderly. Current treatment recommendations are established for young subjects in good general health condition, without taking into account the frailty, comorbidities and polymedications inherent in patients over 75 years old. For locally advanced lower and middle rectal cancers (T3, T4 or N+), these are based on variations of regimens including neoadjuvant chemoradiotherapy, surgery of the rectum with total removal of the mesorectum, and a possibility of adjuvant chemotherapy. This restrictive treatment presents a problem of compliance and is not without adverse effects. Treatment by short exclusive radiotherapy or chemoradiotherapy with close monitoring according to the Watch and Wait strategy can be proposed to fragile patients not eligible for surgery, even if there is a non-negligible risk of recurrence.

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.

Glycosylation status of the membrane protein CD9P-1.

The membrane protein CD9P-1 is a major component of the tetraspanin web, a network of molecular interactions in the plasma membrane, in which it specifically associates with tetraspanins CD9 and CD81. The various functional effects of CD9 and CD81 may be related to their partners. Thus, we have addressed the characterization of the CD9P-1 glycosylation using stably transfected HEK-293 cells. After immunoprecipitation, CD9P-1 was subjected to enzymatic PNGase F cleavage of N-glycans, resulting in Asn to Asp conversion and increase in 1 mass unit. Thus, following protease digestion, deglycosylated peptides were selectively identified by high mass accuracy FTICR-MS, using this conversion as a signature. This has demonstrated that all nine potential N-glycosylation sites were actually engaged. On the other hand, the N-glycan structures were determined combining chemical derivatization and exoglycosidase digestions followed by MALDI-TOF MS, ESI-MS/MS, and GC-MS analysis. CD9P-1 was shown to exhibit more than 40 different N-glycans, essentially composed of complex and high mannose-type structures. Finally, 2-D PAGE and lectino-blot analyses have revealed the presence of at least 17 glycosylated isoforms of CD9P-1 at cell surface. All CD9P-1 isoforms associate with CD9 leading to additional level of complexity of this primary complex in the tetraspanin web.

Membrane microdomains and proteomics: lessons from tetraspanin microdomains and comparison with lipid rafts.

Biological membranes are compartmentalized into microdomains that exhibit particular lipid and protein compositions. Membrane microdomains, such as tetraspanin-enriched microdomains and lipid rafts, have been suggested to play a role in a variety of physiological and pathological processes. Therefore, the characterization of the protein compositions of these microdomains, which is the focus of this review, appears to be a crucial step to better understanding their function. Proteomics has recently allowed the characterization of tetraspanin-enriched microdomains in colon cancer cells. This demonstrated the presence of different categories of membrane proteins and suggested a variation in the composition of tetraspanin-enriched microdomains during tumor progression. On the other hand, proteomics has permitted the identification of hundreds of proteins in lipid rafts of different origins. However, the diversity of methodologies in sample preparation and of strategies in protein identification led to a broad variability in the data obtained. These methodological issues are discussed. Moreover, proteomics has revealed that different sets of proteins were present in tetraspanin-enriched microdomains as compared with lipid rafts, strengthening the idea that these microdomains are distinct structures.

Profiling of the tetraspanin web of human colon cancer cells.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of multimolecular complexes in the plasma membrane. Indeed each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. Thus, tetraspanin-tetraspanin associations lead to a molecular network of interactions, the "tetraspanin web." We performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of three cell lines derived from the primary tumor and two metastases (hepatic and peritoneal) from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9, and the associated proteins were separated by SDS-PAGE and identified by mass spectrometry using LC-MS/MS. This allowed the identification of 32 proteins including adhesion molecules (integrins, proteins with Ig domains, CD44, and epithelial cell adhesion molecule) (EpCAM), membrane proteases (ADAM10, TADG-15, and CD26/dipeptidyl peptidase IV), and signaling proteins (heterotrimeric G proteins). Importantly some components were differentially detected in the tetraspanin web of the three cell lines: the laminin receptor Lutheran/B-cell adhesion molecule (Lu/B-CAM) was expressed only on the primary tumor cells, whereas CD26/dipeptidyl peptidase IV and tetraspanin Co-029 were observed only on metastatic cells. Concerning Co-029, immunohistofluorescence showed a high expression of Co-029 on epithelial cells in normal colon and a lower expression in tumors, whereas heterogeneity in terms of expression level was observed on metastasis. Finally we demonstrated that epithelial cell adhesion molecule and CD9 form a new primary complex in the tetraspanin web.

Proteomic analysis of the tetraspanin web using LC-ESI-MS/MS and MALDI-FTICR-MS.

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.