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Metallothioneins are ubiquitous proteins with a high affinity for heavy metal ions, e.g. zinc, copper and cadmium. Experimentally, metallothionein over-expression in cell lines derived from a variety of cancers has been associated with resistance to anticancer drugs and irradiation therapy. Using a monoclonal antibody (E9) to metallothionein we investigated immunoreactive expression in routinely fixed and paraffin-embedded tissue from 63 cases of malignant melanoma and 13 secondary deposits. Whereas a variety of cells in normal skin showed metallothionein expression, all forms of benign naevi studied were uniformly negative. In contrast 13/30 'thin' (< or = 1.5 mm; 0.7 +/- 0.4), 25/29 'thick' malignant melanoma (> 1.5 mm; 5.5 +/- 3.9) and 12/13 metastases were positive. Six patients with thin and 19 with thick melanoma with metallothionein expression died during a mean observation period of 6.4 +/- 1.8 and 3.6 +/- 2.5 years, respectively, their survival distribution function analyses giving statistically significant results for both the vertical tumour thickness (P < 0.0001) and metallothionein expression (P < 0.0001). These immunohistochemical results, based on routinely processed paraffin-embedded tissue, suggest that metallothionein expression in malignant melanoma is significantly associated with progressive disease and might therefore be a useful prognostic indicator.

The forkhead box transcription factor FOXO3a has recently been identified as central mediator of the cellular response to oxidative stress inducing cell cycle arrest or apoptosis. The aim of our study was to investigate the regulation of FOXO3a in the thyroid and to determine whether alterations in FOXO3a activity occur in thyroid carcinogenesis. In vitro, we demonstrate that FOXO3a activity is negatively regulated by the PI3K/Akt cascade promoting increased phosphorylation and cytoplasmatic accumulation of FOXO3a with decreased transcription of the target genes p27kip (CDKN1B) and Bim (BCL2L11), but increased expression of GADD45A. By contrast, we show that H 2 O 2 exposure activates FOXO3a in thyrocytes with JNK (MAPK8)-mediated nuclear accumulation of FOXO3a and increased expression of the cell cycle arrest genes p27kip and GADD45A. In vivo, we observed a marked cytoplasmatic accumulation of FOXO3a in differentiated thyroid cancers versus an exclusive nuclear accumulation in follicular adenoma and normal thyroid tissue. Moreover, this cytosolic accumulation of FOXO3a correlated with an increased phospho-Akt expression in thyroid malignancies and was accompanied by decreased expression of the FOXO targets p27kip and Bim and an increase in GADD45A mRNA expression in the thyroid cancers. Our data suggest FOXO3a as a novel player of cellular stress response in the thyroid, mediating the thyrocyte's fate either to survive or to undergo apoptosis. Furthermore, PI3K-dependent FOXO3a inactivation may be a novel pathomechanism for the escape from apoptosis in thyroid cancer cells, in particular in follicular thyroid carcinoma.

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