PE
Excitation: 565nm, Emission: 578nm
Epithelial-mesenchymal transition (EMT) is an important process in tumor metastasis. The EMT-related events associated with metastasis of NPC in the absence of EBV have not been elucidated. We established an EBV-negative NPC cell line from a bone marrow biopsy of an NPC patient. Using a Matrigel system we isolated an invasive and non-invasive sublines, designated NPC-BM29 and NPC-BM00. NPC-BM29 acquired an invasive-like phenotype characterized by EMT, marked by down-regulation of E-cadherin and beta-catenin with concomitant increased expression of Ets1. NPC-BM29 cells expressed >or= 10-fold higher of MMP-9 than NPC-BM00 cells. NPC-BM29 cells grew better in 2% serum than NPC-BM00 cells, with a population doubling-time of 26.8 h and 30.7 h, respectively. A marked reduction in colony-formation ability of NPC-BM00 cells compared to NPC-BM29 was observed. Wound-healing assay revealed that NPC-BM29 cells displayed higher motility than NPC-BM00 and the motility was further enhanced by cell treatment with TPA, a PKC activator. Cell surface markers and tumor-associated molecules, AE3, MAK6 and sialyl-Tn, were up-regulated in NPC-BM29 cells, whereas the expression of HLA-DR and CD54 was significantly increased in NPC-BM00 cells. NPC-BM29 consistently released higher levels of IL-8 and IL-10 than NPC-BM00, with low levels of IL-1alpha expression in both cell lines. Higher level of VEGF production was detected in NPC-BM00 than NPC-BM29 cells. These data show that EBV is not required for exhibiting multiple metastatic phenotypes associated with EMT. More studies that target right molecules/signalings associated with the EMT may offer new therapeutic intervention options for NPC invasion and metastasis.
Phenotypic modifications induced by contact allergens on monocyte-derived dendritic cells (MoDC) have been proposed as an in vitro alternative method to discriminate potential sensitizers from irritants. However, the sensitivity of the assay remains controversial. In all the studies reported so far, DC treatment with chemicals was carried out after 5 to 6 days of monocyte culture. Here, we first determined the dynamic range of expression of differentiation and activation markers on human MoDC cultured in the presence, or absence, of TGFbeta. At day three of culture, most monocytes have already differentiated into CD1a+/CD14- DC and, in the presence of TGFbeta, they expressed CD40, CD54 or CD86 antigens with lower fluorescence intensity than 5 day-cultured MoDC. Treatment of 3-day cultured TGFbeta-MoDC with all the tested strong and moderate sensitizers, i.e. NiSO(4), DNCB, balm of Peru, isothiazolinone and cinnamic aldehyde, at non-toxic concentrations, induced significant phenotypic changes, whereas the irritant SLS had no effect. However, a large variability was observed in the number and nature of the modified antigens, according to the chemical and the experiments. This implies that many surface antigens must be analyzed and many experiments carried out to use this assay as an alternative screening method for contact sensitizers.