Loss of E-cadherin leads to upregulation of NFkappaB activity in malignant melanoma.

Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here, we show that loss of E-cadherin leads to induction of nuclear factor kappa B (NFkappaB) activity in melanoma cell lines. Melanoma cells show constitutively active NFkappaB, whereas no activity is found in primary melanocytes. After re-expression of E-cadherin in melanoma cells, strong downregulation of NFkappaB activity was found. Consistently, NFkappaB activity was induced in primary human melanocytes after inhibition of E-cadherin activity by functionally blocking anti-E-cadherin antibodies. Interestingly, re-expression of E-cadherin-blocked p38 MAPK activity and the p38 MAPK inhibitors SB203580 and SB202190 almost completely prevented NFkappaB activation in melanoma cells. Furthermore, cytoplasmatic beta-catenin induced p38 and NFkappaB activation in malignant melanoma. To our knowledge, this is the first report suggesting a correlation between E-cadherin and NFkappaB activity in melanocytes and melanoma cells. In summary, we conclude that loss of E-cadherin and cytoplasmatic beta-catenin induces p38-mediated NFkappaB activation, potentially revealing an important mechanism of tumorigenesis in malignant melanomas.

Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells.

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.

Transcription factors involved in development and progression of malignant melanoma.

Up to date many genes are known to be deregulated in tumor development and progression. Genes important in tumorigenesis belong to families such as proteases, kinases and receptors. However, an important family of proteins is rarely discussed: the mediators of transcriptional control, the transcription factors. Usually, changes in transcription factor expression or activity can lead to more than just one downstream modification, as transcription factors are higher, thinking in a hierarchical way of expression control. In this review we summarize the role of the transcription factors AP-1, AP-2alpha, CREB, CtBP, ETS-1, HMGB1, LEF/TCF/beta-catenin, MITF, NFkappaB, PAX3, SKI, Snail and STAT in carcinogenesis focusing on melanoma development and progression.

The Ets-1 transcription factor is involved in the development and invasion of malignant melanoma.

The Ets-1 transcription factor plays a role in tumor vascularization and invasion by regulating expression of matrix-degrading proteases in endothelial cells and fibroblasts in the tumor stroma. During early embryogenesis, Ets-1 is expressed in migrating neural crest cells from which melanocytes arise. In the present study, we analyzed Ets-1 expression in various melanocytic lesions and investigated its functional importance in malignant melanomas. We found that Ets-1 was upregulated both in vivo and in vitro in malignant melanoma, compared to benign melanocytic lesions and to primary melanocytes. Assessment of DNA-binding and transactivation assays documented a strong Ets activity in melanoma cells. Using an antisense strategy, the expression and activity of Ets-1 were reduced in the melanoma cell line Mel Im. This correlated with a diminished expression of several Ets-1 target genes known to be involved in invasion, such as MMP1, MMP3, uPA and integrin beta3. In line with these findings, the invasive potential of the melanoma cells measured in a Boyden Chamber model was reduced up to 60% after Ets-1 blockade. This can be attributed to the role of Ets-1 in transcriptional regulation of factors involved in invasion of melanoma cells. We conclude that over-expression of Ets-1 during melanoma development contributes to the malignant phenotype.

Loss of E-cadherin expression in melanoma cells involves up-regulation of the transcriptional repressor Snail.

Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression. Here we show that loss of E-cadherin in melanoma cell lines does not involve mutations in the E-cadherin gene, promoter methylation, or alterations in expression of AP-2 transcription factors as suggested previously. In a panel of different melanoma cell lines, E-cadherin expression was negatively regulated by up-regulation of the transcription factor Snail. In comparison with primary human melanocytes, where Snail expression was not detected by reverse transcription-polymerase chain reaction, significant expression was found in all eight melanoma cell lines. In parallel, Western blot and reverse transcription-polymerase chain reaction analysis revealed strong reduction of E-cadherin expression in the melanoma cells. Consistently, transient transfection of a Snail expression plasmid into human primary melanocytes led to significant down-regulation of E-cadherin, whereas transient and stable transfection of an antisense Snail construct induced reexpression of E-cadherin in Mel Ju and Mel Im melanomas. In summary, we conclude that activation of Snail expression plays an important role in down-regulation of E-cadherin and tumorigenesis of malignant melanomas.