Metallothionein expression prevents apoptosis: a study with antisense phosphorothioate oligodeoxynucleotides in a human T cell line.

Human metallothioneins (hMTs), are low molecular weight cysteine-rich proteins that constitute the majority of intracellular protein thiols. Their transcription is regulated by metals, glucocorticoids and cytokines, and in certain tissues it is a highly specialized phenomenon. Although their physiological function is not entirely understood, hMTs induction has been observed to be associated with protection from heavy metal toxicity and cellular resistance to cytotoxic anticancer drugs. However, the main problem in the investigation of the physiological function of hMTs is the absence of any known specific inhibitor, as well as the fact that many genes constitute the hMTs family. As the identification of genes preventing apoptosis is of great interest, we attempted to examine the role of hMTs in the apoptotic process by inhibiting their expression in the immature T cell line CCRF-CEM with antisense sequence-specific phosphorothioate oligodeoxynucleotides (ODNs). In the experimental procedure the cells were activated and cultured in medium containing 20% FBS instead of 10%, during maintenance. We found that the inhibition of hMTs synthesis, induced by the incubation of the cells for 24 hours with ODNs, stimulated the apoptotic process, as confirmed by the characteristic morphological alterations and DNA fragmentation. Quantitative analysis of apoptosis has shown that inhibition of hMTs expression results in a dose-dependent and ODNs sequence-specific induction of apoptosis. Immunocyto-chemical detection of hMTs followed by Tunel assay showed that all the Tunel positive cells were hMTs negative, suggesting that hMTs expression prevents apoptosis. As hMTs induction is rapid and transient in response to stress and/or environmental stimuli, these results indicate that hMTs constitute a cellular protective mechanism, neutralizing external apoptotic signals.