Use of autogenous bone and beta-tricalcium phosphate in maxillary sinus lifting: histomorphometric study and immunohistochemical assessment of RUNX2 and VEGF.

The aim of this study was to compare the use of beta-tricalcium phosphate (ß-TCP) (chronOS) with autogenous bone grafts alone in maxillary sinus elevation surgery. The test samples were ß-TCP alone, ß-TCP mixed with autogenous bone grafts (1:1), and autogenous bone grafts alone. Twelve maxillary sinuses were grafted with ß-TCP (group 1), nine with ß-TCP+autogenous bone graft (group 2), and 12 with autogenous bone graft (group 3). After 6 months, biopsies were obtained concurrent to the placement of dental implants; these were subjected to histomorphometric analysis and immunohistochemical analysis for runt-related transcription factor 2 (RUNX2) and vascular endothelial growth factor (VEGF). The average bone formation in group 1 was 46.3±11.6% in the pristine bone region, 47.6±9.9% in the intermediate region, and 44.8±22.1% in the apical region; in group 2, values were 35.0±15.8%, 32.5±13.7%, and 32.8±16.0%, respectively; in group 3, values were 43.1±16.0%, 31.0±13.0%, and 46.1±16.3%, respectively. Immunostaining of samples in group 2 showed high cellular activity and immature bone; this differed from groups 1 and 3, in which mature bone was demonstrated. Thus, this study showed that ß-TCP presents the same behaviour as autogenous bone graft, which makes it a good bone substitute.

Use of autogenous bone and beta-tricalcium phosphate in maxillary sinus lifting: a prospective, randomized, volumetric computed tomography study.

The correction of bone defects can be performed using autogenous or alloplastic materials, such as beta-tricalcium phosphate (ß-TCP). This study compared the changes in bone volume (CBV) after maxillary sinus lifting using autogenous bone (n = 12), autogenous bone associated with ß-TCP 1:1 (ChronOS; DePuy Synthes, Paoli, CA, USA) (n = 9), and ß-TCP alone (n = 11) as grafting material, by means of cone beam computed tomography (CBCT). CBV was evaluated by comparing CBCT scans obtained in the immediate postoperative period (5-7 days) and at 6 months postoperative in each group using OsiriX software (OsiriX Foundation, Geneva, Switzerland). The results showed an average resorption of 45.7 ± 18.6% for the autogenous bone group, 43.8 ± 18.4% for the autogenous bone+ß-TCP group, and 38.3 ± 16.6% for the ß-TCP group. All bone substitute materials tested in this study presented satisfactory results for maxillary sinus lifting procedures regarding the maintenance of graft volume during the healing phase before the insertion of implants, as assessed by means of CBCT.

Proteomics study of medullary thyroid carcinomas expressing RET germ-line mutations: identification of new signaling elements.

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.

SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.

We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.