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 cytok ines, and in certain tissues it is a highly specialized phenomenon. Al though their physiological function is not entirely understood, hMTs i nduction 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 f unction of hMTs is the absence of any known specific inhibitor, as wel l as the fact that many genes constitute the hMTs family As the identi fication of genes preventing apoptosis is of great interest, we attemp ted 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 th e experimental procedure the cells were activated and cultured in medi um containing 20% FBS instead of 10% during maintenance. We found that the inhibition of hMTs synthesis, induced by the incubation of the ce lls for 24 hours with ODNs, stimulated the apoptotic process,as confir med by the characteristic morphological alterations and DNA fragmentat ion. Quantitative analysis of apoptosis has shown that inhibition of h MTs expression results in a dose-dependent and ODNs sequence-specific induction of apoptosis. Immunocytochemical detection of hMTs followed by Tunel assay showed that all the Tunel positive cells were hMTs nega tive, suggesting that hMTs expression prevents apoptosis. Ar hMTs indu ction is rapid and transient in response to stress and/or environmenta l stimuli, these results indicate that hMTs constitute a cellular prot ective mechanism, neutralizing external apoptotic signals.