Magic™ Anti-BNP monoclonal antibody, clone 36D6

The transwell chamber migration assay and CCD digital camera imaging techniques were used to investigate the relationship between regulatory volume decrease (RVD) and cell migration in nasopharyngeal carcinoma cells (CNE-2Z cells). Both migrated and non-migrated CNE-2Z cells, when swollen by 47% hypotonic solution, exhibited RVD which was inhibited by extracellular application of chloride channel blockers adenosine 5'-triphosphate (ATP), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen. However, RVD rate in migrated CNE-2Z cells was bigger than that of non-migrated cells and the sensitivity of migrated cells to NPPB and tamoxifen was higher than that of non-migrated cells. ATP, NPPB and tamoxifen also inhibited migration of CNE-2Z cells. The inhibition of migration was positively correlated to the blockage of RVD, with a correlation coefficient (r) = 0.99, suggesting a functional relationship between RVD and cell migration. We conclude that RVD is involved in cell migration and RVD may play an important role in migratory process in CNE-2Z cells.

SHOX and SHOX2 transcription factors are highly homologous, with even identical homeodomains. Genetic alterations in SHOX result in two skeletal dysplasias; Leri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), while no human genetic disease has been linked to date with SHOX2. SHOX2 is, though, involved in skeletal development, as shown by different knockout mice models. Due to the high homology between SHOX and SHOX2, and their functional redundancy during heart development, we postulated that SHOX2 might have the same transcriptional targets and cofactors as SHOX in limb development. We selected two SHOX transcription targets regulated by different mechanisms: 1) the natriuretic peptide precursor B gene (NPPB) involved in the endochondral ossification signalling and directly activated by SHOX; and 2) Aggrecan (ACAN), a major component of cartilage extracellular matrix, regulated by the cooperation of SHOX with the SOX trio (SOX5, SOX6 and SOX9) via the protein interaction between SOX5/SOX6 and SHOX. Using the luciferase assay we have demonstrated that SHOX2, like SHOX, regulates NPPB directly whilst activates ACAN via its cooperation with the SOX trio. Subsequently, we have identified and characterized the protein domains implicated in the SHOX2 dimerization and also its protein interaction with SOX5/SOX6 and SHOX using the yeast-two hybrid and co-immunoprecipitation assays. Immunohistochemistry of human fetal growth plates from different time points demonstrated that SHOX2 is coexpressed with SHOX and the members of the SOX trio. Despite these findings, no mutation was identified in SHOX2 in a cohort of 83 LWD patients with no known molecular defect, suggesting that SHOX2 alterations do not cause LWD. In conclusion, our work has identified the first cofactors and two new transcription targets of SHOX2 in limb development, and we hypothesize a time-and tissue-specific functional redundancy between SHOX and SHOX2.